Delivery of RNA therapeutics using cyclic prodrug nucleic acids

Abstract

The present invention provides oligonucleotides referred to as cyclic prodrug nucleic acids ("CPNs") as described herein, compositions comprising the same, and methods of using the same. This design of cyclic prodrug nucleic acids maintains its cyclic form until the cyclic domain is cleaved in situ by RNase H or Dicer or other intracellular factors.

Description

[Technical Field]

[0001] Related applications This application claims the benefits of U.S. Provisional Patent Application No. 63 / 453,909, filed on 22 March 2023. All teachings of the said application are incorporated herein by reference. [Background technology]

[0002] The processing and translation of targeted RNA can be regulated by multiple mechanisms using antisense oligonucleotides. These include cleavage of targeted RNA by RNase H, regulation of abnormal splicing, increased processing and translation of targeted RNA, and inhibition of translation due to steric hindrance. Targeted RNA can be mRNA or non-coding RNA. In other mechanisms, the antisense strand of a double-stranded siRNA can be incorporated into the AGO, inhibiting translation by the siRNA mechanism. In other mechanisms, such as adenosine deaminase (ADAR)-based or CRISPR-based models acting on RNA, antisense can edit RNA or DNA, thereby regulating translation and processing.

[0003] Over the years, it has been understood that antisense hybridization and affinity influence the selectivity for targeted RNA. Furthermore, for antisense nucleic acids to be used as drugs, nuclease stability provided by nucleotide binding, such as modification of phosphorothioates, is crucial.

[0004] Assuming that nuclease stability is crucial for survival and potency within the intracellular compartment, and given that antisense degradation is shown to occur from the 3' end, the focus was on modifying the 3' end to slow degradation. These designs included capping the 3' end, forming a hairpin loop at the 3' end, creating oligos with secondary structures containing a 3'-3' bond, or affixing two antisense oligonucleotides to the 3' end. These types of antisense showed increased nuclease stability, but did not improve antisense potency. Unfortunately, these modifications also increased the inflammatory response, thereby limiting the therapeutic index.

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

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

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

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

[0009] The present invention provides a structural class of oligonucleotides referred herein as “cyclic prodrug nucleic acids” (CPNs) or equivalently “cyclic prodrug oligos” (CPOs). A CPN comprises a “functional domain” and a “cyclization domain.”

[0010] The "functional domain" contains oligonucleotides that provide function to the CPN.

[0011] In various embodiments, the "cyclic 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 (directly or via a linker segment), and the 3' end of the oligonucleotide of the functional domain is linked to the second nucleic acid molecule; the first and second nucleic acid molecules are independently 6 to 30 nucleotides long, the nucleotides of the first and second nucleic acid molecules are independently selected from RNA, DNA, or a combination of RNA and DNA, the first and second nucleic acid molecules are complementary to each other, have opposite polarities, and hybridize to form a double-stranded portion (e.g., Figure 1).

[0012] In various embodiments, the "cyclic 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 (directly or via a linker segment), and the 3' end of the oligonucleotide of the functional domain is linked to the second nucleic acid molecule; the first and second nucleic acid molecules are independently 6 to 30 nucleotides long, and the nucleotides of the first and second nucleic acid molecules are independently selected from RNA, DNA, or a combination of RNA and DNA (provided that neither the first nor the second nucleic acid molecule is DNA); the first and second nucleic acid molecules are complementary to each other, have opposite polarities, and hybridize to form a double-stranded portion (e.g., Figure 1).

[0013] Oligonucleotides of the functional domain and first and second nucleic acid molecules of the cyclic domain are further described herein.

[0014] The CPN of this invention adopts an intramolecular cyclic structure for complementarity between the first and second nucleic acid molecules of the cyclic domain that forms an intramolecular double helix. This intramolecular double helix formation alters the shape of the functional domain and the accessibility of the 5' and 3' ends of the oligonucleotides of the functional domain. This structure combines key attributes to create optimal nucleic acid-based therapeutics. In CPN, the nucleotides of the oligonucleotides of the functional domain do not participate in the formation of the intramolecular double helix.

[0015] Improving RNA therapeutics involves improving the delivery of nucleic acids to the cytoplasm and nucleus in a stable form, thereby improving the efficacy of RNA therapeutics. The 3' and 5' ends of nucleic acids bind to pattern recognition receptors and exonucleases, thereby influencing the availability of RNA therapeutics. Therefore, delivering RNA therapeutics to the appropriate compartments by preventing the ends from accessing these factors would help improve RNA therapeutics. Conditional release of RNA therapeutics into cells by naturally occurring factors allows the drug to become available in the desired compartment.

[0016] In gene and RNA expression regulation, this structure masks the 5' end of the oligonucleotide functional domain, thereby reducing interaction with PRRs and enabling endosomal escape. The CPN structure also masks the 3' end, providing nuclease stability. Upon entering the cytoplasm or nucleus, the double-stranded portion formed by the first and second nucleic acid molecules is cleaved by RNase H, Dicer, restriction enzymes, or other intracellular factors, opening the cyclic structure and thereby presenting the oligonucleotide of the functional domain, enabling it to perform its function. This process of cleaving the cyclic domain of the CPN to present the oligonucleotide of the functional domain is referred to herein as "in situ activation."

[0017] In various embodiments, upon cleavage, the first and second nucleic acid molecules of the cyclic domain are unable to hybridize, thereby preventing the re-cyclic formation of the oligonucleotide of the functional domain.

[0018] When CPNs are cyclic, they may exhibit fewer exposed phosphorothioate bonds or fewer PS bonds, potentially leading to fewer polyanion-related side effects known to occur with PS-oligonucleotides (e.g., complement activation and prolongation of partial thromboplastin time). Additionally, cyclic CPNs have reduced protein binding, decreased off-target interactions with non-targeted RNAs, and increased nuclease stability and endosomal evasion.

[0019] The CPN according to the present invention can be prepared using standard techniques for synthesizing constituent oligonucleotides and is useful for all purposes where functional oligonucleotides and nucleic acids are useful.

[0020] The foregoing merely summarizes certain aspects of the present invention and is not intended to limit, nor should it be construed as limiting, the invention. All patents, patent applications, and other publications listed herein are incorporated herein by reference in their entirety.

[0021] The aforementioned and other objects, features and advantages of the present invention will become apparent from the following more detailed description of preferred embodiments of the invention, as shown in the accompanying drawings. In the accompanying drawings, similar reference numerals throughout the different figures refer to the same parts. The drawings are not necessarily to scale and instead focus on illustrating the principles of the present invention. [Brief explanation of the drawing]

[0022] [Figure 1]Figures 1A to 1H show various embodiments of the cyclic prodrug oligonucleotide (100) according to the present invention. A solid line (101) represents the functional domain. A dashed line (102) represents the first nucleic acid molecule of the cyclic domain. A dashed line (103) represents the second nucleic acid molecule of the cyclic domain. L represents the linkage between the functional domain and the cyclic domain, either directly or via a linker. The cyclic prodrug oligonucleotide maintains its cyclic form until the double-stranded region formed by the first and second nucleic acid molecules is cleaved in situ by an RNase H or Dicer, restriction enzyme, or other intracellular factor, releasing the functional domain. An optional linker L may be present on either or both of the first or second nucleic acid molecules of the cyclic domain, as long as the linker does not impede the ability of the first or second nucleic acid molecules to hybridize to each other or to be cleaved in situ. [Figure 2] Figures 2A–2J show a comparison of antisense oligonucleotides in cyclized prodrug form that target apolipoprotein C-III (APOC3). Data are plotted as a percentage of control cells (cells reverse-transfected with the vehicle alone). Gapmer: 5'-GCTTCTTGTCCAGCTTTATT-3' (SEQ ID NO: 33). Gapmer-untargeted control: 5'-CCAAATCTTATAATAACTAC-3' (SEQ ID NO: 36). [Figure 3] Figures 3A–3D show a comparison between gapmer-type antisense oligonucleotides and cyclized prodrugs targeting microtubule-associated protein tau (MAPT). Data are plotted as a percentage of control cells (cells reverse-transfected with the vehicle alone). Gapmer control: 5'-CCGTTTTCTTACCACCCT-3' (SEQ ID NO: 37). [Modes for carrying out the invention]

[0023] Please understand that both the general explanation above and the detailed explanation below are illustrative and descriptive, and not limiting.

[0024] In this specification, unless otherwise specified, the use of the singular form includes the plural form. Where used herein, the use of "or" means "and / or" unless otherwise specified. Furthermore, the use of the term "including" and other forms such as "includes" and "included" is not limited. Also, terms such as "element" or "component" include both elements and components containing one unit and elements and components containing two or more subunits, unless otherwise specified.

[0025] Section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein. All documents or parts of documents cited herein, including but not limited to patents, patent applications, articles, books, and papers, are expressly incorporated herein by reference with respect to the parts and all of the documents described herein.

[0026] Features may be described herein as part of the same or different aspects or embodiments of the present invention for clarity and concise explanation. Those skilled in the art will understand that the scope of the present invention may include embodiments having all or some combinations of the features described herein as part of the same or different embodiments.

[0027] Through this disclosure, various aspects of the present invention can be presented in range form. It should be understood that the range form description is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the present invention. Therefore, the range description should be considered to specifically disclose all possible subranges and increments and / or parts within that range, as well as the individual numerical values ​​within that range. For example, a range description such as 6-30 should be considered to include subranges such as 6-27, 6-25, 6-15, 8-20, 8-15, 10-15, as well as the individual numbers having that range, e.g., 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, where applicable, between numbers (e.g., one-tenth, one-hundredth, etc.) or any part or increment of those numbers. This applies regardless of the width of the range.

[0028] As used herein, the term “about” is to be understood by those skilled in the art and varies to some extent depending on the context in which it is used. As used herein when referring to measurable values ​​such as quantities or temporal durations, the term “about” means to include variations of ±20% or ±10%, including ±5%, ±1%, and ±0.1% from the specified value, such variations being suitable for carrying out the disclosed method.

[0029] The present invention also provides oligonucleotides called cyclic prodrug nucleic acids ("CPNs"). A CPN comprises a "functional domain" and a "cyclization domain".

[0030] The "functional domain" contains oligonucleotides that provide function to the CPN. The cyclic domain includes a first nucleic acid molecule and a second nucleic acid molecule, the 5' end of the oligonucleotide of the functional domain is linked to the first nucleic acid molecule directly or via a linker segment, and the 3' end of the oligonucleotide of the functional domain is linked to the second nucleic acid molecule; the first and second nucleic acid molecules are independently 6 to 30 nucleotides long, the nucleotides of the first and second nucleic acid molecules are independently selected from RNA, DNA, or a combination of RNA and DNA, the first and second nucleic acid molecules are complementary and opposite polarities to each other, and hybridize to form a double-stranded portion (e.g., Figures 1A to 1C). In various embodiments, the first and second nucleic acid molecules are not both DNA.

[0031] In various embodiments, the CPN of the present invention includes a structure according to formula I or formula II: 3'-Y n Y6Y5Y4Y2Y2Y1-5'-5'-Functional Domain Oligonucleotide-3'-5'-X1X2X3X4X5X6X m -3' Equation I 5'-Y1Y2Y3Y4Y5Y6Y n -3'-5'-Functional Domain Oligonucleotide-3'-3'-X m X6X5X4X3X2X1-5' Formula II During the ceremony Y1~Y n is the first nucleic acid molecule as defined herein; X1~X m is the second nucleic acid as defined herein; n is 0 to 44; and m is between 0 and 44. A functional domain oligonucleotide is any oligonucleotide as defined herein.

[0032] In various embodiments, the CPN of the present invention includes a structure according to formula III, IV, V, VI, or VII: 3'-Yn Y6Y5Y4Y2Y2Y1-5'-L-5'-Functional domain oligonucleotide-3'-5'-X1X2X3X4X5X6X m -3' Formula III 5'-Y1Y2Y3Y4Y5Y6Y n -3'-L-5'-Functional domain oligonucleotide-3'-3'-X m X6X5X4X3X2X1-5' Formula IV 3'-Y n Y6Y5Y4Y2Y2Y1-5'-5'-Functional domain oligonucleotide-3'-L-5'-X1X2X3X4X5X6X m -3' Formula V 5'-Y1Y...​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​In some embodiments, the first nucleic acid molecule is bonded to the 5' end of the functional domain via a 5'-5' linkage, and the second nucleic acid molecule is bonded to the 3' end of the oligonucleotide via a 3'-5' linkage. In other embodiments, the first nucleic acid molecule is bonded to the 5' end of the functional domain via a 5'-3' linkage, and the second nucleic acid molecule is bonded to the 3' end of the oligonucleotide via a 3'-3' linkage.

[0034] In either configuration, the first and second nucleic acid molecules of the cyclic domain are complementary and opposite in polarity, allowing the first nucleic acid molecule to hybridize with the second, thereby forming a cyclic structure (e.g., Figures 1A-1C). This design of the cyclic prodrug nucleic acid maintains its cyclic form until the double-stranded region is cleaved by intracellular factors such as RNase H and Dicer. This structure allows for increased stability, specificity, and release of the functional domain in the appropriate compartment.

[0035] Oligonucleotides of a functional domain provide the desired function to the CPN. For example, for gene expression regulation, oligonucleotides of a functional domain are complementary to the targeted RNA. As used herein, the terms “oligonucleotide of a functional domain” and “functional domain” are used interchangeably.

[0036] The CPN of the present invention adopts an intramolecular cyclic structure for complementarity between the first nucleic acid molecule and the second nucleic acid molecule in the cyclic domain that forms the intramolecular double helix. The nucleotides of the oligonucleotides in the functional domain do not participate in the formation of the intramolecular double helix.

[0037] As those skilled in the art will recognize, despite the requirement that the nucleotides of the functional domain oligonucleotide are not involved in the formation of the intramolecular double helix in the CPN of the present invention, one or more nucleotides at the 5' and / or 3' ends of the functional domain oligonucleotide can be designed to be involved in the formation of a double helix resulting in a cyclic shape, insofar as the functional domain oligonucleotide can still play its role in the cleavage of the CPN double helix and the resulting linearization of the oligonucleotide compound.

[0038] The nucleotides of the first and second nucleic acid molecules in the cyclic domain are not part of the oligonucleotides of the functional domain and therefore do not provide the desired function to the CPN.

[0039] In various embodiments, the oligonucleotide of the functional domain is DNA, RNA, or a combination thereof.

[0040] In various embodiments, the first nucleic acid molecule of the circular domain is DNA, and the second nucleic acid molecule is RNA. In various embodiments, the first nucleic acid molecule of the circular domain is RNA, and the second nucleic acid molecule is DNA. In various embodiments, the first nucleic acid molecule of the circular domain is RNA, and the second nucleic acid molecule is also RNA. In various embodiments, the first nucleic acid molecule of the circular domain is DNA, and the second nucleic acid molecule is also DNA. In various embodiments, the first and second nucleic acid molecules of the circular domain are not both DNA.

[0041] In various embodiments, the nucleotides of the first nucleic acid molecule and / or the second nucleic acid molecule of the cyclic domain are a combination of RNA and DNA.

[0042] In various embodiments, the oligonucleotides of the functional domain are unmodified.

[0043] In various embodiments, the first and second nucleic acid molecules of the cyclic domain are unmodified.

[0044] "Unmodified" means that the nucleotides and / or the first and second nucleic acid molecules of the functional domain oligonucleotide are intended to contain naturally occurring nucleic acid bases, sugars, and internucleotide skeletons.

[0045] In some embodiments, at least one nucleotide of the functional domain oligonucleotide is modified. In some embodiments, two or more nucleotides of the functional domain oligonucleotide are modified. In some embodiments, at least half of the nucleotides of the functional domain oligonucleotide are modified. In some embodiments, all of the nucleotides of the functional domain oligonucleotide are modified.

[0046] In various embodiments, the nucleotides of the functional domain oligonucleotide include modifications of internucleotide bonds, sugars, heterocyclic bases, or combinations thereof. These modifications can also be appropriately positioned at specific locations within the functional domain oligonucleotide. Other chemistry and modifications are readily available in accordance with this disclosure and are known in the field of oligonucleotides, encompassed by the term “modified” as used herein in the context of oligonucleotides.

[0047] Unless otherwise specified herein, the functional domain comprises oligonucleotides 15 to 500 nucleotides in length. In some embodiments, the oligonucleotides of the functional domain are 17 and 300 nucleotides in length. In some embodiments, the oligonucleotides of the functional domain are 17 and 200 nucleotides in length. In some embodiments, the oligonucleotides of the functional domain are 17 and 100 nucleotides in length. In some embodiments, the oligonucleotides of the functional domain are 17 and 50 nucleotides in length. In some embodiments, the oligonucleotides of the functional domain are 17 and 25 nucleotides in length.

[0048] In various embodiments, the oligonucleotides of the functional domain are 50 and 250 nucleotides long. In various embodiments, the oligonucleotides of the functional domain are 50 and 150 nucleotides long.

[0049] In various embodiments, the functional domain contains oligonucleotides 15 to 50 nucleotides long. In various embodiments, the functional domain contains oligonucleotides 17 to 40 nucleotides long. In various embodiments, the functional domain contains oligonucleotides 17 to 25 nucleotides long. In various embodiments, the oligonucleotides of the functional domain are 17 and 22 nucleotides long.

[0050] In various embodiments, the oligonucleotides of the functional domain are 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides long. In various embodiments, the functional domain is 17 nucleotides long. In various embodiments, the functional domain is 18 nucleotides long. In various embodiments, the functional domain is 19 nucleotides long. In various embodiments, the functional domain is 20 nucleotides long. In various embodiments, the functional domain is 21 nucleotides long. In various embodiments, the functional domain is 22 nucleotides long. In various embodiments, the functional domain is 23 nucleotides long. In various embodiments, the functional domain is 24 nucleotides long. In various embodiments, the functional domain is 25 nucleotides long. In various embodiments, the functional domain is 26 nucleotides long. In various embodiments, the functional domain is 27 nucleotides long. In various embodiments, the functional domain is 28 nucleotides long. In various embodiments, the functional domain is 29 nucleotides long. In various embodiments, the functional domain is 30 nucleotides long. In various embodiments, the functional domain is 31 nucleotides long. In various embodiments, the functional domain is 32 nucleotides long. In various embodiments, the functional domain is 33 nucleotides long. In various embodiments, the functional domain is 34 nucleotides long. In various embodiments, the functional domain is 35 nucleotides long. In various embodiments, the functional domain is 36 nucleotides long. In various embodiments, the functional domain is 37 nucleotides long. In various embodiments, the functional domain is 38 nucleotides long. In various embodiments, the functional domain is 39 nucleotides long. In various embodiments, the functional domain is 40 nucleotides long.

[0051] In various embodiments, the functional domain includes, but is not limited to, an oligonucleotide selected from antisense oligonucleotides, microRNAs (miRNAs), siRNAs, piRNAs, hnRNAs, ncRNAs, snRNAs, miRNA mimes, sgRNAs, esiRNAs, shRNAs, lncRNAs, mRNAs, CRISPR-based systems, RNA-acting adenosine deaminase (ADAR) systems, or splicing oligonucleotides.

[0052] In some embodiments, the oligonucleotide of the functional domain may be an aptamer.

[0053] In some embodiments, the functional domain may be an adeno-associated virus (AAV).

[0054] In various embodiments, the functional domain includes, but is not limited to, an oligonucleotide selected from immunostimulatory oligonucleotides or immunoinhibitory oligonucleotides (also called immunoantagonist oligonucleotides).

[0055] The only limitation on the nucleotide and internucleotide bonding of functional domain oligonucleotides is that they do not preclude the functional domain from performing its intended function (for example, in the case of a functional domain that is an antisense oligonucleotide, hybridizing with a complementary RNA segment under physiological conditions to form a double helix, which is a substrate for RNase H). Preferred nucleotide and internucleotide bonding enhances the stability of the CPN against nuclease and other forms of chemical degradation, and / or enhances the functional domain's ability to perform its intended function.

[0056] In various embodiments, the nucleotide-nucleotide bonds of the functional domain are phosphorothioate nucleotide bonds, phosphodiester nucleotide bonds, or a combination thereof.

[0057] In various embodiments, the nucleotides of the first and second nucleic acid molecules of the cyclic domain include modifications of internucleotide bonds, sugars, heterocyclic bases, or combinations thereof. These modifications can also be positioned at specific locations within the first and second nucleic acid molecules of the cyclic domain.

[0058] As used herein, the terms “first nucleic acid molecule and second nucleic acid molecule of the cyclic domain” or “cyclic domain” are interchangeable.

[0059] In various embodiments, the nucleotide linkage between the first and second nucleic acid molecules of the cyclic domain is a phosphorothioate nucleotide linkage, a phosphodiester nucleotide linkage, or a combination thereof. In various embodiments, the nucleotide linkage between the first and second nucleic acid molecules of the cyclic domain may be modified as known to those skilled in the art, provided that the cyclic domain remains a substrate for RNase H or Dicer or other intracellular proteins. In various embodiments, the nucleotide linkage of the cyclic domain is a phosphodiester nucleotide linkage.

[0060] In various embodiments, the first nucleic acid molecule and the second nucleic acid molecule are independently DNA or RNA. In various embodiments, the first nucleic acid molecule is DNA and the second nucleic acid molecule is RNA. In various embodiments, the first nucleic acid molecule is RNA and the second nucleic acid molecule is DNA. In various embodiments, the first nucleic acid molecule is RNA and the second nucleic acid molecule is RNA.

[0061] In various embodiments, the first nucleic acid molecule is DNA, and the second nucleic acid molecule is DNA.

[0062] In various embodiments, the nucleotides of the first nucleic acid molecule and / or the second nucleic acid molecule are a combination of DNA and RNA. In various embodiments, the first nucleic acid molecule is DNA, and the second nucleic acid molecule is a combination of RNA and DNA. In various embodiments, the first nucleic acid molecule is a combination of RNA and DNA, and the second nucleic acid molecule is DNA. In various embodiments, the first nucleic acid molecule is RNA, and the second nucleic acid molecule is a combination of RNA and DNA. In various embodiments, the first nucleic acid molecule is a combination of RNA and DNA, and the second nucleic acid molecule is RNA. In various embodiments, the first nucleic acid molecule is a combination of RNA and DNA, and the second nucleic acid molecule is a combination of RNA and DNA.

[0063] In various embodiments, the RNA and / or DNA of the first nucleic acid molecule and the second nucleic acid molecule are unmodified. In various embodiments, the first nucleic acid molecule and the second nucleic acid molecule each independently contain an oligonucleotide of 6 to 50 nucleotides in length. In various embodiments, the first nucleic acid molecule and the second nucleic acid molecule of the cyclic domain each independently contain an oligonucleotide of 6 to 25 nucleotides in length. In various embodiments, the first nucleic acid molecule and the second nucleic acid molecule each independently contain an oligonucleotide of 6 to 12 nucleotides in length. In various embodiments, the first nucleic acid molecule and the second nucleic acid molecule each independently contain an oligonucleotide of 6 to 10 nucleotides in length. In various embodiments, the first nucleic acid molecule and the second nucleic acid molecule each independently contain an oligonucleotide of 6 to 8 nucleotides in length.

[0064] In various embodiments, the first nucleic acid molecule and the second nucleic acid molecule independently contain an oligonucleotide with a length of 6 to 14 nucleotides.

[0065] In various embodiments, the first nucleic acid molecule and the second nucleic acid molecule of the cyclic domain are independently 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 long.

[0066] In some embodiments, the first nucleic acid molecule and the second nucleic acid molecule are of the same length. In some embodiments, the first nucleic acid molecule and the second nucleic acid molecule are of different lengths.

[0067] As used herein, the term “polarity” refers to the concept of orientation in the primary structure (e.g., 3'→5' and 5'→3' for DNA and RNA, or N-terminus→C-terminus (or vice versa) for PNA). If the CPN of the present invention comprises, for example, a first nucleic acid molecule and a second nucleic acid molecule that hybridize in an antiparallel manner by Watson-Crick base pairing, the first nucleic acid molecule may have a 5'→3' (or 2') configuration, and the nucleotide sequence to which it is complementary in the second nucleic acid molecule may have a 3' (or 2')→5' configuration. Alternatively, the first nucleic acid molecule may have a 3' (or 2')→5' configuration, and the nucleotide sequence to which it is complementary in the second nucleic acid molecule may have a 5'→3' (or 2') configuration.

[0068] The specific internal bonding between the first and second nucleic acid molecules is not important, as long as these two segments of the cyclic domain hybridize.

[0069] In one embodiment, the 5' end of the oligonucleotide of the functional domain is linked to the 5' end of the first nucleic acid molecule via a 5'-5' bond, and the 3' end of the oligonucleotide of the functional domain is linked to the 5' end of the second nucleic acid molecule via a 3'-5' bond.

[0070] In one embodiment, the 5' end of the oligonucleotide of the functional domain and the 3' end of the first nucleic acid molecule are linked via a 5'-3' bond, and the 3' end of the oligonucleotide of the functional domain and the 3' end of the second nucleic acid molecule are linked via a 3'-3' bond.

[0071] In various embodiments, the first nucleic acid molecule is at least 95% complementary to the second nucleic acid molecule. In various embodiments, the first nucleic acid molecule is at least 97% complementary to the second nucleic acid molecule. In various embodiments, the first nucleic acid molecule is at least 98% complementary to the second nucleic acid molecule. In various embodiments, the first nucleic acid molecule is at least 99% complementary to the chain of the second nucleic acid molecule. In various embodiments, the first nucleic acid molecule is at least 100% complementary to the second nucleic acid molecule.

[0072] In various embodiments, an oligonucleotide of a functional domain is linked to a first nucleic acid molecule and / or a second nucleic acid molecule via a linker segment. In various embodiments, the linker segment is directly linked, a nucleotide or oligonucleotide of 2-5 nucleotides in length, or other chemical moieties, or a combination thereof. In some embodiments, the linker segment may be cleavable.

[0073] In various embodiments, the linker segment between the oligonucleotide and the first nucleic acid molecule is the same as the linker segment between the oligonucleotide and the second nucleic acid molecule. In various embodiments, the linker segment is a direct bond.

[0074] In various embodiments, the linker segment between the oligonucleotide and the first nucleic acid molecule is different from the linker segment between the oligonucleotide and the second nucleic acid molecule.

[0075] The only limitation of the linker segment is that it does not preclude the essential functions of the CPN, namely (a) the CPN's ability to form an intramolecular cyclic structure under desired conditions (e.g., physiological conditions) and (b) the ability of the functional domain to perform its intended function.

[0076] 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) n NH- (where n is 2, 3, 4, 5, or 6). Alternatively, the linker segment can be a combination of the above.

[0077] In a preferred embodiment, the linker is a direct bond.

[0078] In one embodiment, the linker is ethylene glycol. In embodiments, the linker is C2-C6 alkyl. In embodiments, the linker is C2 alkyl. In embodiments, the linker is C3 alkyl. In embodiments, the linker is C4 alkyl. In embodiments, the linker is C5 alkyl. In embodiments, the linker is C6 alkyl.

[0079] In one embodiment, the linker is selected from lipids, fatty acids, antibodies, galnac, peptides, or proteins.

[0080] When all bases in at least one strand of a nucleic acid pair are found on the opposite side of their complementary base pairs, such a strand is considered to be completely complementary to its sequence in the other strand. When one or more bases of such a strand are found at positions opposite any other base other than its complementary base pair, that base is considered a "mismatch" and the strand is considered to be partially complementary. Thus, a strand can be partially complementary (e.g., 0% < x < 100% complementary) to varying degrees until the bases no longer align (at which point they are non-complementary (e.g., 0% complementary)). As will be readily understood and recognized by those skilled in the art, perfect (i.e., complete, 100%) complementarity is not required for hybridization of nucleic acid strands (e.g., oligonucleotides, antisense, or others).

[0081] Examples of first and second nucleic acid molecules with cyclic domains useful for CPN oligonucleotides described herein include, but are not limited to, the nucleic acids listed in Table 2. [Table 1]

[0082] Uppercase C / G / T / A represent DNA with phosphorothioate bonds; lowercase c / g / t / a represent RNA with phosphodiester bonds; C1 / G1 / T1 / A1 represent DNA with phosphodiester bonds; lowercase c1 / g1 / t1 / a1 represent RNA with phosphodiester bonds.

[0083] In various embodiments, the cyclic prodrug nucleic acid according to the present invention is part of a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0084] A pharmaceutical composition comprising the cyclic prodrug nucleic acid of the present invention may further comprise any other agents or treatments useful for treating or preventing a disease or symptom, without impairing the function of the cyclic prodrug nucleic acid according to the present invention. Agents useful for treating or preventing a disease or symptom include, but are not limited to, small molecules, peptides, vaccines, antigens, antibodies, preferably monoclonal antibodies, cytotoxic agents, kinase inhibitors, allergens, antibiotics, siRNA molecules, antisense oligonucleotides, TLR antagonists (e.g., TLR3 and / or TLR7 antagonists and / or TLR8 antagonists and / or TLR9 antagonists), chemotherapeutic agents (both traditional chemotherapy and modern targeted therapies), targeted therapeutic agents, activated cells, peptides, proteins, gene therapy vectors, peptide vaccines, protein vaccines, DNA vaccines, adjuvants, and costimulatory molecules (e.g., cytokines, chemokines, protein ligands, transactivators, peptides comprising peptides or modified amino acids), or combinations thereof. Alternatively, the cyclic prodrug nucleic acid according to the present invention may be administered in combination with other compounds (for example, formulated with lipids or liposomes, or conjugated to peptides, antibodies, or small molecules) to enhance the specificity or magnitude of gene expression regulation of the cyclic prodrug nucleic acid according to the present invention.

[0085] Functional domain In any of the embodiments described herein, the functional domain of CPN may be an oligonucleotide as further described below. The first and second nucleic acid molecules of the cyclic domain and linker segment of CPN are as described above unless otherwise specified.

[0086] As described above, the functional domain includes, but is not limited to, oligonucleotides selected from antisense oligonucleotides, microRNAs (miRNAs), siRNAs, piRNAs, hnRNAs, ncRNAs, snRNAs, miRNA mimes, sgRNAs, esiRNAs, shRNAs, lncRNAs, mRNAs, aptamers, CRISPR-based systems, RNA-acting adenosine deaminase (ADAR) systems, or splicing oligonucleotides. In various embodiments, the functional domain includes, but is not limited to, oligonucleotides selected from immunostimulatory oligonucleotides or immunoinhibitory oligonucleotides (also known as immune antagonist oligonucleotides).

[0087] In embodiments in which the functional domain oligonucleotide is an oligonucleotide of a CRISPR-based system or an RNA-acting adenosine deaminase (ADAR) system, the portion of the oligonucleotide complementary to the target RNA (complementary domain) is at least 90% complementary to the portion of the target RNA over its entire length, preferably at least 95%, preferably at least 97%, preferably at least 98%, preferably at least 99%, or preferably at least 100% complementary.

[0088] In various embodiments, the oligonucleotide of the functional domain contains at least one phosphorothioate internucleotide bond. In various embodiments, at least half of the internucleotide bond is phosphorothioate. In various embodiments, all of the internucleotide bond is phosphorothioate.

[0089] In various embodiments, the oligonucleotide of the functional domain is single-stranded.

[0090] In various embodiments, the functional domain oligonucleotide is at least 90% complementary to a portion of the target RNA over its entire length. In various embodiments, the functional domain oligonucleotide is at least 95% complementary to a portion of the target RNA over its entire length. In various embodiments, the functional domain oligonucleotide is at least 97% complementary to a portion of the target RNA over its entire length. In various embodiments, the functional domain oligonucleotide is at least 98% complementary to a portion of the target RNA over its entire length. In various embodiments, the functional domain oligonucleotide is at least 99% complementary to a portion of the target RNA over its entire length. In various embodiments, the functional domain oligonucleotide is at least 100% complementary to a portion of the target RNA over its entire length.

[0091] In some embodiments, the target RNA may be mRNA, pre-mRNA, ncRNA, lncRNA, or microRNA. In some embodiments, the target RNA is mRNA.

[0092] Examples of gene-regulating oligonucleotides suitable for use in the functional domain of the cyclic prodrug nucleic acid of the present invention include, but are not limited to, the gene-regulating oligonucleotides listed in Table 1. Functional domain oligonucleotides directed to any other sequence within the target RNA or to any other target RNA of interest are well within the scope of the art. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7]

[0093] Uppercase G / C / A / T - DNA with phosphorothioate links; lowercase a / t / c / g - RNA; G1 / C1 / A1 / U1 - 2'OME or 2'MOE ribonucleotides; A2 / T2 / C2 / G2 - DNA with phosphodiester links; lowercase a1 / t1 / c1 / g1 - RNA with phosphodiester links; G3 / C3 / A3 / U3 - 2'MOE ribonucleotides with phosphorothioate links; G4 / C4 / A4 / T4 / U4 - 2'MOE phosphodiester links

[0094] Inhibition of gene expression In various embodiments, the present invention provides cyclic prodrug nucleic acids (CPNs) comprising a functional domain containing gene-regulating oligonucleotides (i.e., oligonucleotides capable of regulating the expression of a target gene). Such oligonucleotides may include, but are not limited to, antisense oligonucleotides, microRNAs (miRNAs), miRNA mimes, piRNAs, hnRNAs, ncRNAs, siRNAs, snRNAs, sgRNAs, esiRNAs, shRNAs, or lncRNAs.

[0095] In various embodiments, the present invention provides a cyclic prodrug nucleic acid (CPN) comprising an oligonucleotide 15 to 45 nucleotides in length and containing a functional domain complementary to a target RNA; the 5' end of the oligonucleotide is linked (directly or via a linker segment) to a first nucleic acid molecule, and the 3' end of the oligonucleotide is linked to a second nucleic acid molecule; the first and second nucleic acid molecules form the cyclic domain described herein; and the oligonucleotide of the functional domain includes a gene regulatory oligonucleotide. In various embodiments, the oligonucleotide of the functional domain is modified. In various embodiments, the first and second nucleic acid molecules are independently 6 to 30 nucleotides in length, the nucleotides of the first and second nucleic acid molecules are independently selected from RNA or DNA, the first and second nucleic acid molecules are complementary to each other, have opposite polarities, and hybridize to form a double-stranded portion. In various embodiments, the first and second nucleic acid molecules are not both DNA.

[0096] In various embodiments, the modification of the oligonucleotide includes at least one modified nucleic acid base, sugar, and / or internucleotide bond.

[0097] As shown herein, the CPN of the present invention, comprising a gene-regulating oligonucleotide as a functional domain, surprisingly exhibited increased potency. Furthermore, the gene-regulating oligonucleotide of the CPN lacks a free 5' end, and the CPN may have lower inflammatory properties. This design allows the antisense oligonucleotide to unfold into a linear structure and be active upon in-situ activation in cells expressing the target RNA.

[0098] In various embodiments, if the functional domain of the CPN is an antisense oligonucleotide, it is part of a cyclic prodrug until it is present in the cytoplasm or nucleus, where the cyclic domain (composed of a first nucleic acid molecule and a second nucleic acid molecule) is cleaved by RNase H or Dicer, thereby linearizing the CPN and allowing the antisense oligonucleotide of the functional domain to bind to the target RNA. The change from cyclic to linear morphology has been confirmed by RNase H and / or Dicer cleavage studies. In the linear morphology, the functional domain hybridizes with a complementary target RNA (at least under physiological conditions) to form a double helix. Depending on the intended mechanism of action of the functional domain, the double helix formed with the target RNA will result in translational regulation. For example, this double helix is ​​a substrate of RNase H, and in the presence of RNase H and under appropriate (e.g., physiological) conditions, the target RNA strand of the double helix is ​​cleaved by RNase H, thereby inhibiting expression.

[0099] CPNs containing antisense oligonucleotides as functional domains maintain their activity in cell cultures. The expected advantages of these CPNs are reduced interaction with non-target macromolecules (including nucleic acids and proteins) due to the formation of an intramolecular cyclic structure, thereby reducing polyanion-related side effects. Furthermore, due to the cyclic structure, these CPNs can escape endosomes due to the lack of a free 5' end of the oligonucleotide, which is incompatible with interaction with pattern recognition receptors.

[0100] The oligonucleotides of the present invention are isolated oligonucleotides. The term “isolated” means modified or removed from their natural state by human intervention. For example, oligonucleotides naturally present in living animals are not “isolated,” but synthetic oligonucleotides, or oligonucleotides partially or completely separated from their naturally occurring coexisting substances, are “isolated.” Isolated oligonucleotides may exist in a substantially purified form or in a non-native environment, such as a cell to which the oligonucleotide is delivered. The oligonucleotides of the present invention may include partially purified DNA and / or RNA, substantially pure DNA and / or RNA, synthetic DNA and / or RNA, or recombinantly produced DNA and / or RNA, as well as modified DNA and / or RNA that differs from naturally occurring DNA and / or RNA by the addition, deletion, substitution and / or modification of one or more nucleotides. Such modifications may include the addition of non-nucleotide material to the terminal of the oligonucleotide or to one or more internal nucleotides of the oligonucleotide, including modifications that make the oligonucleotide resistant to nuclease digestion.

[0101] The terms "microRNA," "miRNA," and "MiR" are interchangeable and refer to endogenous or artificial non-coding RNAs that can regulate gene expression. miRNAs are thought to function through RNA interference. The design of such microRNAs is within the scope of the art of the art.

[0102] The terms "piRNA" and "Piwi-interacting RNA" are interchangeable and refer to a class of small RNA molecules involved in gene silencing. PiRNA molecules are typically 26–31 nucleotides long. The design of such piRNAs is within the scope of the art of the art.

[0103] Splitmer In various embodiments, functional domain antisense oligonucleotides are as described in International Publication No. 2020 / 191177, which is incorporated in whole herein by reference. In various embodiments, functional domain antisense oligonucleotides are modified oligonucleotides comprising or consisting of an antisense oligonucleotide compound 17 to 25 nucleotides in length, the antisense oligonucleotide compound comprising a 3' domain and a 5' domain adjacent to the 3' domain, the 3' domain beginning with a 3' terminal nucleotide and being 10 to 12 nucleotides in length, each nucleotide comprising a deoxyribonucleotide and a phosphodiester or phosphorothioate nucleotide interbond or combination thereof; the 5' domain beginning with the first nucleotide following the 3' domain and extending to a 5' terminal nucleotide, the 5' domain comprising an unmodified deoxyribonucleotide, an unmodified ribonucleotide, a modified deoxyribonucleotide, a modified ribonucleotide, or a combination thereof, wherein the 5' domain comprises at least three modified deoxyribonucleotides or modified ribonucleotides. The modified deoxyribonucleotides and / or modified ribonucleotides in the 5' domain do not need to be consecutive. The modified deoxyribonucleotides and / or modified ribonucleotides in the 5' domain prevent RNase H cleavage in the 5' domain. In various embodiments, the modified deoxyribonucleotide or modified ribonucleotide includes a modified base, a modified sugar, and / or a modified skeleton. In various embodiments, the modified deoxyribonucleotide or modified ribonucleotide includes a modified sugar and / or a modified skeleton.

[0104] In some embodiments, at least half of the nucleotides in the 5' domain comprise a modified deoxyribonucleotide or modified ribonucleotide containing a modified sugar and / or backbone. Such antisense oligonucleotides are referred to as “splitmers.”

[0105] In some embodiments, all nucleotides in the 5' domain consist of modified deoxyribonucleotides or modified ribonucleotides containing a modified sugar and / or backbone.

[0106] If fewer than all nucleotides in the 5' domain are modified, the modified and unmodified nucleotides are arranged such that there are no more than two adjacent unmodified nucleotides in the 5' domain.

[0107] In various embodiments, the functional domain splitmer comprises 17 to 25 ligated nucleotides having at least 12 consecutive nucleic acid bases complementary to equal-length portions of the target RNA.

[0108] In some embodiments, the modified ribonucleotide of the splitmer comprises a 2'-substituted nucleotide as described herein. In some embodiments, the 2'-substituted nucleotide is selected from 2'O-methyl ribonucleoside (2'-OME) or 2'-methoxyethyl ribonucleoside (2'-MOE).

[0109] In some embodiments, the 3' domain includes 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 includes 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 includes nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 from the 3' end.

[0110] In some embodiments, when the 3' domain of the antisense oligonucleotide is 12 nucleotides long, the antisense oligonucleotide of the present invention is of formula (I): 5'-N m N 14 N 13 N 12 N 11 N 10 N9N8N7N6N5N4N3N2N1-3' It is expressed by, in the formula, N is any nucleotide; N 13 ~N m This includes the 5' domain; N1~N 12 This includes the 3' domain; and m is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

[0111] In some embodiments, when the 3' domain of the antisense oligonucleotide is 11 nucleotides long, the antisense oligonucleotide of the present invention is of formula (Ia): 5'-N m N 14 N 13 N 12 N 11 N 10 N9N8N7N6N5N4N3N2N1-3' It is expressed by, in the formula, N is any nucleotide; N 12 ~N m This includes the 5' domain; N1~N 11 This includes the 3' domain; and m is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

[0112] In some embodiments, the antisense oligonucleotide of the present invention is of formula (Ib): 5'-N m N 14 N 13 N 12 N 11 N 10 N9N8N7N6N5N4N3N2N1-3' It is expressed by, in the formula, N is any nucleotide; N 11 ~N m This includes the 5' domain; N1~N 10 This includes the 3' domain; and m is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

[0113] 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.

[0114] In various embodiments, the functional domain splitmer antisense oligonucleotide compound is 17 to 25 nucleotides long and contains at least 12 consecutive nucleic acid bases complementary to an equal-length portion of the target RNA sequence, the antisense oligonucleotide compound comprises a 3' domain and a 5' domain adjacent to the 3' domain, the 3' domain being 10 to 12 nucleotides long and beginning with a 3' terminal nucleotide, each nucleotide containing a deoxyribonucleotide and a phosphodiester or phosphorothioate nucleotide internucleotide bond or a combination thereof; the 5' domain is adjacent to the 3' domain The 5' domain begins with the first nucleotide following the 'in' and continues to the terminal nucleotide at the 5' end, and the 5' domain comprises an unmodified deoxyribonucleotide, an unmodified ribonucleotide, a modified deoxyribonucleotide, a modified ribonucleotide, or a combination thereof, provided that the 5' domain comprises at least three modified deoxyribonucleotides or modified ribonucleotides, and the modified deoxyribonucleotides and / or modified ribonucleotides in the 5' domain do not need to be consecutive; the modified deoxyribonucleotides and / or modified ribonucleotides in the 5' domain prevent RNase H cleavage in the 5' domain. In various embodiments, the modified deoxyribonucleotide or modified ribonucleotide comprises a modified base, a modified sugar, and / or a modified skeleton. In various embodiments, the modified deoxyribonucleotide or modified ribonucleotide comprises a modified sugar, and / or a modified skeleton.

[0115] In some embodiments, at least half of the nucleotides in the 5' domain comprise a modified deoxyribonucleotide or modified ribonucleotide containing a modified sugar and / or backbone.

[0116] In some embodiments, all nucleotides in the 5' domain consist of modified deoxyribonucleotides or modified ribonucleotides containing a modified sugar and / or backbone.

[0117] If fewer than all nucleotides in the 5' domain are modified, the modified and unmodified nucleotides are arranged such that there are no more than two adjacent unmodified nucleotides in the 5' domain.

[0118] In some embodiments, the 3' domain is 12 nucleotides long and contains nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 from the 3' end (position 1 is the 3' end). In some embodiments, the 3' domain is 11 nucleotides long and contains nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 from the 3' end. In some embodiments, the 3' domain is 12 nucleotides long and contains nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 from the 3' end.

[0119] In some embodiments, the nucleotides of the 3' domain contain native nucleic acid bases. In some embodiments, the nucleic acid bases and sugars of the nucleotides of the 3' domain of the antisense oligonucleotide according to the present invention are unmodified. In this respect, the nucleic acid bases and sugars of the nucleotides of the 3' domain of the antisense oligonucleotide according to the present invention are naturally occurring. Each of the nucleotides of the 3' domain contains a deoxyribonucleotide and a phosphodiester or phosphorothioate internucleotide bond or a combination thereof. The nucleotides of the 3' domain contain a native deoxyribose sugar and a phosphorothioate, phosphodiester or other phosphorus bond or a combination thereof, which are known to activate RNase H.

[0120] In various embodiments, at least one nucleotide of the 3' domain contains a modified nucleic acid base.

[0121] In various embodiments, the 9th or 10th nucleotide from the 3' end is not modified. In various embodiments, the 9th and 10th nucleotides from the 3' end are not modified. In various embodiments, the 11th nucleotide from the 3' end is not modified. In various embodiments, the 9th, 10th, and 11th nucleotides from the 3' end are not modified. In various embodiments, the 12th nucleotide from the 3' end is not modified. In various embodiments, the 9th, 10th, 11th, and 12th nucleotides from the 3' end are not modified.

[0122] In some embodiments, the oligonucleotide contains at least one phosphorothioate internucleotide bond. In some embodiments, at least half of the internucleotide bond is phosphorothioate. In some embodiments, all of the internucleotide bond is phosphorothioate. In some embodiments, at least half of the internucleotide bond is phosphodiester. In some embodiments, all of the internucleotide bond is phosphodiester.

[0123] In various embodiments, the antisense oligonucleotide is single-stranded.

[0124] As used herein, the term “5' domain” refers to the nucleotides beginning with the first nucleotide following the 3' domain and extending toward the 5' end. The 5' domain hybridizes to the target RNA, but RNase H cannot cleave the target RNA within this domain. The term “5' domain” is generally 2 to 15 nucleotides long and, depending on the length of the 3' domain, refers to the 11th to 25th nucleotides (the 1st nucleotide being the 3' end), the 12th to 25th nucleotides, or the 13th to 25th nucleotides of an antisense oligonucleotide measured from the 3' end. In some embodiments, the 5' domain refers to the nucleotides from the 11th to the 5' terminal nucleotide of the functional domain splitmer oligonucleotide (the 1st nucleotide being the 3' terminal nucleotide). In some embodiments, the 5' domain refers to the nucleotides from the 12th to the 5' terminal nucleotide of the functional domain splitmer oligonucleotide. In some embodiments, the 5' domain refers to the nucleotides from the 13th to the 5' terminal nucleotide of the functional domain splitmer oligonucleotide.

[0125] For example, an antisense oligonucleotide compound with a length of 20 nucleotides may contain a 3' domain from position 1 to position 12 and a 5' domain from position 13 to position 20. The designation of modified nucleotides is position-specific, not nucleotide-specific.

[0126] The 5' domain contains a nucleotide having a non-RNase H activating modification, such as a modified sugar and / or a modified skeleton that does not activate RNase H. In some embodiments, the 5' domain contains a nucleotide containing a modified sugar. In some embodiments, the 5' domain contains a nucleotide containing a modified skeleton. In some embodiments, the 5' domain contains a nucleotide containing both a modified sugar and a modified skeleton. In various embodiments, the modified skeleton is a non-phosphorus skeleton.

[0127] This antisense design allows for targeted RNA cleavage at a specific site toward the 5' end of the 3' domain.

[0128] In any of these embodiments, at least half of the nucleotides in the 5' domain are intended to include modifications or substitutions of the skeleton and / or modifications or substitutions of sugars. In some embodiments, all nucleotides at positions within the 5' domain include modifications or substitutions of the skeleton and / or modifications or substitutions of sugars. In one embodiment, the 5' domain includes at least four nucleotides containing the modified skeleton and / or sugars. In one embodiment, the 5' domain includes at least five nucleotides containing the modified skeleton and / or sugars. In one embodiment, the 5' domain includes at least six nucleotides containing the modified skeleton and / or sugars. In one embodiment, the 5' domain includes at least seven nucleotides containing the modified skeleton and / or sugars. In one embodiment, the 5' domain includes at least eight nucleotides containing the modified skeleton and / or sugars. In one embodiment, all of the nucleotides in the 5' domain are nucleotides containing the modified skeleton and / or sugars.

[0129] The embodiments discussed herein are particularly intended to be implemented with respect to the position relative to the 3' end in the context of specific nucleotides and positions. For example, an antisense oligonucleotide having a modified nucleotide at position 13 refers to an antisense oligonucleotide having a modified nucleotide at position 13 from the 3' end of the antisense oligonucleotide.

[0130] In various embodiments, the functional domain antisense oligonucleotide is at least 90% complementary to a portion of the target RNA throughout its entire length.

[0131] Gapmar In various embodiments, the functional domain oligonucleotide is a “gapmer.” As used herein, a gapmer is a chimeric antisense oligonucleotide containing a central block of deoxynucleotide monomers of sufficient length to induce RNase H cleavage. Typically, the gapmers of the present invention are for one or more mRNAs encoding a target mRNA. The design of such gapmers is within the realm of the art.

[0132] In various embodiments, the functional domain antisense oligonucleotide is a modified oligonucleotide comprising or comprising a region having a gapmer motif defined by two outer regions or "wings" and a central or inner region or "gap". The three regions of the gapmer motif (5'-wing, gap, and 3'-wing) form a contiguous sequence of nucleosides in which at least a portion of the sugar moieties of the nucleosides of each wing are different from at least a portion of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing closest to the gap (the most 3'-nucleosides of the 5'-wing and the most 5'-nucleosides of the 3'-wing) are different from the sugar moieties of the adjacent gap nucleosides, and thus define the boundary between the wing and the gap (i.e., the wing / gap junction). In certain embodiments, the sugar moieties within the gap are identical to each other. In certain embodiments, the gap comprises one or more nucleosides having sugar moieties different from the sugar moieties of one or more other nucleosides within the gap. In certain embodiments, the sugar motifs of the two wings are identical to each other (symmetric gapmer). In certain embodiments, the sugar motif of the 5'-wing is different from the sugar motif of the 3'-wing (asymmetric gapmer).

[0133] In certain embodiments, the gapmer wings independently contain 1 to 6 nucleosides. In certain embodiments, the gapmer wings independently contain 1 to 5 nucleosides. In certain embodiments, the gapmer wings contain the same number of nucleosides. In certain embodiments, the gapmer wings contain 4 nucleosides. In certain embodiments, each nucleoside in each wing of the gapmer is a modified nucleoside.

[0134] In certain embodiments, the gapmer contains 7 to 24 nucleosides. In certain embodiments, the gapmer contains 7 to 18 nucleosides. In certain embodiments, the gapmer contains 9 to 14 nucleosides. In certain embodiments, the gapmer contains 7 to 23 nucleosides. In certain embodiments, the gapmer contains 9 nucleosides. In certain embodiments, the gapmer contains 10 nucleosides. In certain embodiments, the gapmer contains 11 nucleosides. In certain embodiments, the gapmer contains 13 nucleosides. In certain embodiments, the gapmer contains 14 nucleosides. In certain embodiments, the gapmer contains 17 nucleosides. In certain embodiments, the gapmer contains 18 nucleosides. In certain embodiments, each nucleoside in the gap of the gapmer is an unmodified 2'-deoxynucleoside.

[0135] In certain embodiments, the gapmer is a deoxygapmer. In various embodiments, the gap-side nucleoside of each wing / gap junction is an unmodified 2'-deoxynucleoside, and the wing-side nucleoside of each wing / gap junction is a modified nucleoside. In certain embodiments, each nucleoside of the gap is an unmodified 2'-deoxynucleoside. In certain embodiments, each nucleoside of each wing of the gapmer is a modified nucleoside.

[0136] Here, the lengths (number of nucleosides) of the three regions of the gapmer can be given using the notation [5'-number of nucleosides in the wing]-[number of nucleosides in the gap]-[3'-number of nucleosides in the wing]. Thus, a 5-10-5 gapmer consists of 5 linked nucleosides in each wing and 10 linked nucleosides in the gap. If a particular modification follows such nomenclature, the modification is a modification of the wing, and the gap nucleosides contain unmodified deoxynucleoside 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.

[0137] In certain embodiments, the modified oligonucleotide is a 4-13-4 MOE or OMe gapmer. In certain embodiments, the modified oligonucleotide is a 5-11-5 MOE or OME gapmer. In certain embodiments, the modified oligonucleotide is a 3-15-3 BNA gapmer. In certain embodiments, the modified oligonucleotide is a 3-15-3 LNA gapmer.

[0138] In any embodiment described herein, a modified oligonucleotide comprises or consists of a region having a completely modified sugar motif. In such embodiments, each nucleoside in the completely modified region of the modified oligonucleotide contains a modified sugar moiety. In certain embodiments, each nucleoside in the entire modified oligonucleotide contains a modified sugar moiety. In certain embodiments, a modified oligonucleotide comprises or consists of a region having a completely modified sugar motif, and each nucleoside within the completely modified region contains the same modified sugar moiety, referred herein as a uniformly modified sugar motif. In certain embodiments, a completely modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each uniformly modified nucleoside contains the same 2'-modification. In certain embodiments, a uniformly modified sugar motif is 12 to 30 nucleosides long. In certain embodiments, each nucleoside in a uniformly modified sugar motif is a 2'-substituted nucleoside, a sugar substitute, or a bicyclic nucleoside. In certain embodiments, each nucleoside of a uniformly modified sugar motif contains either a 2'-OCH2CH2OCH3 group or a 2'-OCH3 group. In certain embodiments, a modified oligonucleotide having at least one completely modified sugar motif may also have at least one, at least two, at least three, or at least four 2'-deoxynucleosides.

[0139] Examples of cyclic prodrug nucleic acids useful for gene silencing include, but are not limited to, the cyclic prodrug nucleic acids listed in Table 3. Cyclic prodrug nucleic acids having functional domains directed to any other target of interest are well within the realm of the art. All internucleotide bonds are phosphorothioate bonds unless otherwise specified. The sequence numbers of the oligonucleotides of the cyclic and functional domains of the CPN compounds shown below can be found in Tables 1 and 2 above. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0140] Uppercase G / C / A / T - DNA with phosphorothioate links; lowercase a / t / c / g - RNA with phosphodiester links; G1 / C1 / A1 / U1 - 2'OME or 2'MOE ribonucleotides; A2 / T2 / C2 / G2 - DNA with phosphodiester links; lowercase a1 / t1 / c1 / g1 - RNA with phosphodiester links; G3 / C3 / A3 / U3 - 2'MOE ribonucleotides with phosphorothioate links

[0141] Splicing oligonucleotides. In various embodiments, the present invention provides cyclic prodrug nucleic acids (CPNs) comprising a functional domain containing a splicing oligonucleotide. In various embodiments, the oligonucleotide of the functional domain is modified.

[0142] As used herein, the term “splicing oligonucleotide” refers to an antisense oligonucleotide for modulating splicing. For splice modulation, antisense oligonucleotides bind to target RNA, modulate splicing, and thereby regulate protein expression. The cyclic structure allows for reduced protein binding, reduced polyanionic features, and lack of terminal accessibility, which allows for endosomal escape and reduces interaction with pattern recognition receptors.

[0143] In various embodiments, the oligonucleotide for regulating splicing is snRNA. The terms “snRNA” and “nuclear small RNA” are interchangeable and refer to a class of small RNAs involved in various processes, including RNA splicing and transcription factor regulation. Subclasses of nucleolar small RNA (snoRNA) are also included. This term is also intended to include artificial snRNAs, such as antisense derivatives of snRNA. The design of such snRNAs is within the scope of the art of the art.

[0144] In various embodiments, the functional domain oligonucleotide is as described in International Publication No. 2021 / 055011, which is incorporated herein in its entirety by reference. Specifically, in various embodiments, the functional domain oligonucleotide comprises an oligonucleotide containing 14 to 30 linked nucleotides complementary to a target pre-mRNA containing retained introns, the antisense oligonucleotide comprising 1 to 3 DNA regions, each region independently containing 2 to 5 consecutive deoxyribonucleotides, and the remaining nucleotides being 2'-substituted, nonionic, or constrained sugar nucleotides, or a combination thereof. In various embodiments, the 2'-substituted nucleotide is selected from 2'O-methylribonucleoside or 2'-methoxyethylribonucleoside (MOE).

[0145] A CPN having a functional domain containing an oligonucleotide that modulates splicing is useful for selecting a first mRNA transcript in a gene containing at least two mRNA transcripts, wherein the oligonucleotide contains at least 12 consecutive nucleic acid bases complementary to equal-length portions of the target pre-mRNA; the oligonucleotide targets the splice site of the pre-mRNA for the second mRNA transcript, thereby blocking the splice site for the second mRNA transcript and directing the splicing of the pre-mRNA to the first mRNA transcript.

[0146] In some embodiments, the splicing oligonucleotide comprises one region containing 2 to 5 consecutive deoxyribonucleotides, with the remaining nucleotides being 2'-substituted, nonionic, or constrained sugar nucleotides, or a combination thereof. In some embodiments, the splicing oligonucleotide comprises two regions independently containing 2 to 5 consecutive deoxyribonucleotides, with the remaining nucleotides being 2'-substituted, nonionic, or constrained sugar nucleotides, or a combination thereof. In some embodiments, the splice comprises three regions independently containing 2 to 5 consecutive deoxyribonucleotides, with the remaining nucleotides being 2'-substituted, nonionic, or constrained sugar nucleotides, or a combination thereof. In some embodiments, the consecutive deoxyribonucleotides are 2 to 4 nucleotides long. In some embodiments, the consecutive deoxyribonucleotides are 4 nucleotides long.

[0147] In various embodiments, the region of consecutive deoxyribonucleotides in the splicing oligonucleotide is located at the 5' end of the antisense oligonucleotide, at the 3' end of the splicing oligonucleotide, or flanked by 2'-substituted, nonionic, or constrained sugar nucleotides, or a combination thereof. In various embodiments, the consecutive deoxyribonucleotides are located at the 5' end of the splicing oligonucleotide. In various embodiments, the consecutive deoxyribonucleotides are located at the 3' end of the splicing oligonucleotide. In various embodiments, the consecutive deoxyribonucleotides are flanked by 2'-substituted, nonionic, or constrained sugar nucleotides, or a combination thereof.

[0148] In various embodiments, the splicing oligonucleotide of a functional domain comprises 14 to 30 linked nucleotides having at least 12 consecutive nucleic acid bases complementary to equal-length portions of the target pre-mRNA containing retained introns, and the antisense oligonucleotide comprises 1 to 3 DNA regions, each independently containing 2 to 5 consecutive deoxyribonucleotides, with the remaining nucleotides being 2'-substituted, nonionic, or constrained sugar nucleotides, or a combination thereof.

[0149] In various embodiments, the 2'-substituted nucleotide is as described herein. In various embodiments, the 2'-substituted nucleotide is selected from 2'O-methylribonucleotide or 2'-MOE.

[0150] In various embodiments, the functional domain oligonucleotide comprises a single region containing 2 to 5 consecutive deoxyribonucleotides. In various embodiments, the consecutive deoxyribonucleotides are located at the 5' end of the splicing oligonucleotide, at the 3' end of the antisense oligonucleotide, and are flanked by 2'-substituted, nonionic, or constrained sugar nucleotides or combinations thereof. In various embodiments, the consecutive deoxyribonucleotides are located at the 5' end of the splicing oligonucleotide. In various embodiments, the consecutive deoxyribonucleotides are located at the 3' end of the splicing oligonucleotide.

[0151] In some embodiments, the consecutive deoxyribonucleotides are 2 to 4 nucleotides long. In some embodiments, the consecutive deoxyribonucleotides are 4 nucleotides long.

[0152] In some embodiments, the exon is adjacent to the 5' splice region of the retained intron. In some embodiments, the exon is adjacent to the 3' splice region of the retained intron. In some embodiments, the exon is adjacent to the 5' splice region of the retained intron, and the exon is adjacent to the 3' splice region of the retained intron.

[0153] ADAR In various embodiments, the present invention provides cyclic prodrug nucleic acids (CPNs) comprising a functional domain containing an antisense oligonucleotide of an adenosine deaminase (ADAR) system acting on RNA. ADARs are a group of enzymes that catalyze the conversion of adenosine (A) to inosine (I) in a process known as RNA editing. While ADARs can act on various types of RNA, editing events in the coding region of mRNA are of particular interest because the base pair of I is similar to that of guanosine (G). Thus, all A-to-I changes catalyzed by ADARs are read as A-to-G changes during translation, potentially altering the sequence and function of proteins. This re-coding ability makes ADARs an attractive therapeutic tool for correcting gene mutations within mRNA.

[0154] The recognition domain recognizes and binds to specific double-stranded RNA (dsRNA), and the catalytic domain converts adenosine in the target dsRNA to inosine by deamination. In RNA, inosine functions similarly to guanosine for translation and replication, for example, due to its similarity to guanosine. Inosine hybridizes and / or binds to cytosine, but prior to the deamination of adenosine to inosine, the corresponding nucleotide pair is thymine. Furthermore, inosine has been found to mimic guanosine for translational purposes in most cases. Therefore, the conversion of adenosine to inosine in mRNA can result in codon changes that can alter the encoded protein and its function. There are three known ADAR proteins expressed in humans: ADAR1, ADAR2, and ADAR3. ADAR1 and ADAR2 are expressed throughout the body, while ADAR3 is expressed only in the brain.

[0155] ADAR proteins are proteins that are naturally expressed in various cells, tissues, organs, and / or organisms. Several ADAR proteins, such as ADAR1 and ADAR2, have been reported to be able to edit adenosine by deamination, converting adenosine to inosine, and providing several functions, including being read as G or similarly to G during translation. Mechanisms of ADAR-mediated mRNA editing (e.g., deamination) have been reported. For example, ADAR proteins have been reported to catalyze the conversion of adenosine to inosine on mismatched double-stranded RNA substrates. As will be recognized by those skilled in the art, inosine can be recognized as guanosine by cellular translation and / or splicing mechanisms. Therefore, ADARs can be used for functional adenosine-to-guanosine editing of nucleic acids, such as pre-mRNA and mRNA substrates.

[0156] Without being bound by any theory or hypothesis, ADAR-mobilizing RNA ("arRNA") recognizes and binds to target nucleic acids, such as target adenosine in RNA, for ADAR-mediated editing of the target adenosine. The arRNA acts by hybridizing to its target RNA in a sequence-specific manner to form a double-stranded RNA, which then mobilizes adenosine deaminase (ADAR) that acts on RNA to deaminate the target adenosine in the target RNA.

[0157] ADAR-mediated RNA editing can offer several advantages over DNA editing, for example, simplified delivery because it does not require the expression of recombinant proteins such as Cas9. Since both ADAR1 and ADAR2 are endogenous enzymes, arRNA-only cell delivery may be sufficient for editing. Any off-target effects, if any, are transient because there is no change in genomic DNA. Furthermore, ADAR-mediated editing can be used in postmittal cells and does not require an HDR template for repair. Three vertebrate ADAR genes with common functional domains have been reported (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 three ADARs contain a dsRNA-binding domain (dsRBD) that can contact a dsRNA substrate. Some ADAR1s also contain a Z-DNA-binding domain. ADAR1 has been reported to be significantly expressed in the brain, lungs, kidneys, liver, and heart, and this can occur in two isoforms. In some embodiments, isoform p150 can be induced by interferon, while isoform p110 can be constitutively expressed. In some embodiments, p110 has been reported to be ubiquitous and constitutively expressed, so utilizing p110 may be beneficial. ADAR2 can be highly expressed, for example, in the brain and lungs, and has been reported to be localized only to the nucleus. ADAR3 is catalytically inactive and has been reported to be expressed only in the brain. Potential differences in tissue expression can be taken into consideration when selecting therapeutic targets.

[0158] The use of oligonucleotides for RNA editing by ADARs has been reported. In particular, this disclosure recognizes that previously reported techniques generally have one or more drawbacks, such as low stability (e.g., oligonucleotides with natural RNA sugars), low editing efficiency, low editing specificity (e.g., some As are edited in a portion of the target nucleic acid that is substantially complementary to the oligonucleotide), specific structures in the oligonucleotide for ADAR recognition / recruitment, and exogenous proteins (e.g., proteins engineered to recognize oligonucleotides (e.g., using target nucleic acids) that have a specific structure and / or double helix for editing).

[0159] In some embodiments, the provided CPN-arRNA may direct the correction of a guanosine (G) to adenosine (A) mutation in a target sequence or its product. In some embodiments, the correction of the G to A mutation is or includes the conversion of A to inosine (I), which can be read as G during translation or other biological processes. In some embodiments, the provided CPN-arRNA may direct the correction of a G to A mutation in a target sequence or its product via ADAR-mediated deamination. In some embodiments, the provided CPN-arRNA may direct the correction of a G to A mutation in a target sequence or its product via ADAR-mediated deamination by recruiting endogenous ADARs (e.g., in target cells) and promoting ADAR-mediated deamination. However, nevertheless, this disclosure is not limited to any particular mechanism. In some embodiments, the present disclosure provides CPN-arRNA, compositions, methods, etc., that can operate via double-stranded RNA interference, single-stranded RNA interference, RNase H-mediated knockdown, steric hindrance of translation, ADAR-mediated deamination, or a combination of two or more such mechanisms.

[0160] In various embodiments, CPN-arRNA can hybridize to a target RNA sequence nucleic acid at any stage of RNA processing, including but not limited to pre-mRNA or mature mRNA. In some embodiments, CPN-arRNA can hybridize to any element of an oligonucleotide nucleic acid or its complement, including but not limited to a promoter region, enhancer region, transcription stop region, translation start signal, translation stop signal, coding region, non-coding region, exon, intron, intron / exon or exon / intron junction, 5'UTR, or 3'UTR. In some embodiments, CPN-arRNA hybridizes to two or more variants of a transcript derived from the sense strand of the target site (e.g., target sequence).

[0161] In some embodiments, when the CPN-arRNA or composition is brought into contact with a target nucleic acid containing target adenosine in a system (e.g., an ADAR-mediated deamination system), the modification of target adenosine (e.g., deamidation of target A) is improved compared to that observed under reference conditions (e.g., selected from the group consisting of the absence of the composition, the presence of a reference oligonucleotide or the composition, and combinations thereof).

[0162] As will be recognized by those skilled in the art, structural features of the present disclosure, such as modifications of nucleic acid bases, modifications of sugars, modifications of internucleotide bonds, binding phosphorus stereochemistry, and combinations thereof, can be utilized with various suitable base sequences to provide CPN-arRNAs and compositions having desired properties and / or activities. For example, a CPN-arRNA for adenosine modification (e.g., conversion to I in the presence of ADAR protein) typically has a sequence that is sufficiently complementary to the sequence of the target nucleic acid containing the target adenosine. In many embodiments, for example, to target a G to A mutation, a CPN-arRNA may selectively target one and only target adenosine for modifications that convert to I by ADAR, for example.

[0163] The nucleotide sequences of CPN-arRNAs are typically of sufficient length and complementarity to their target nucleic acids, such as RNA transcripts (e.g., pre-mRNA, mature mRNA, etc.), for site-directed editing of target adenosine, as will be recognized to those skilled in the art. In some embodiments, the arRNA of the CPN-arRNA is complementary to a portion of the target RNA sequence containing the target adenosine (as will be recognized to those skilled in the art, in many cases the target nucleic acid is longer than the arRNA of the CPN-arRNA of this disclosure, and the complementarity can be appropriately assessed based on the shorter of the two).

[0164] As will be recognized by those skilled in the art, the CPN-arRNAs of the present invention can be used to improve oligonucleotides in the prior art (for example, described in International Publication Nos. 2016097212, 2017220751, 2018041973, 2018134301A1, 2021071858, and 2022 / 091100, where each oligonucleotide and oligonucleotide composition is incorporated independently by reference). In some embodiments, the disclosure provides improvements to the prior art by applying the CPN-arRNAs described herein to previously reported oligonucleotide sequences. In some embodiments, the disclosure provides CPN-arRNA compositions of previously reported oligonucleotides that may be useful for adenosine editing. In some embodiments, the disclosure provides improvements to previously reported adenosine-edited oligonucleotide compositions by performing such editing using the CPN-arRNA compositions.

[0165] In some embodiments, CPN-arRNA can form a dsRNA structure with a target mRNA via base pairing. In some embodiments, the formed dsRNA structure (optionally having a secondary mismatch) contains a bulge that can promote ADAR binding and thus ADAR-mediated editing (e.g., deamination of target adenosine). Various techniques can be used to evaluate / characterize the CPN-arRNA according to this disclosure.

[0166] In some embodiments, the functional domain oligonucleotide is an antisense oligonucleotide that recruits an endogenous ADAR (RNA-acting adenosine deaminase) enzyme to edit an endogenous transcript.

[0167] The oligonucleotide of the ADAR system itself contains one or more domains. One domain, known as the complementary domain, contains a region of consecutive nucleotides complementary to the target RNA. In various embodiments, the complementary domain is about 15 to about 120 nucleotides long. In various embodiments, the complementary domain is about 17 to about 60 nucleotides long. Another domain, known as the mobilization domain, contains a region of oligonucleotide that mobilizes the ADAR enzyme.

[0168] In embodiments where the functional domain of CPN is an antisense oligonucleotide of the ADAR system, the lengths of the first nucleic acid molecule and the second nucleic acid molecule can be increased. For example, the first nucleic acid molecule and the second nucleic acid molecule each independently contain an oligonucleotide of 6 to 50 nucleotides in length. In some embodiments, the first nucleic acid molecule and the second nucleic acid molecule of the cyclic domain each independently contain an oligonucleotide of 6 to 25 nucleotides in length. In some embodiments, the first nucleic acid molecule and the second nucleic acid molecule each independently contain an oligonucleotide of 6 to 12 nucleotides in length. In some embodiments, the first nucleic acid molecule and the second nucleic acid molecule each independently contain an oligonucleotide of 6 to 10 nucleotides in length. In some embodiments, the first nucleic acid molecule and the second nucleic acid molecule each independently contain an oligonucleotide of 6 to 8 nucleotides in length.

[0169] CRISPR In various embodiments, the present invention provides a cyclic prodrug nucleic acid (CPN) comprising a functional domain containing an antisense oligonucleotide (e.g., guide RNA) for a CRISPR-based system.

[0170] The CRISPR / Cas system ("CRISPR system") uses short RNA molecules (e.g., guide RNA) to recognize specific DNA targets and recruit Cas enzymes to those specific DNA targets.

[0171] Generally, the CRISPR system refers collectively to transcripts and other elements that are involved in or direct the activity of CRISPR-related ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activated CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (including "direct repeats" and tracrRNA processing partial direct repeats in the context of endogenous CRISPR systems), guide sequences (guide RNA), or other sequences and transcripts from the CRISPR locus. In some embodiments, one or more elements of the CRISPR system are derived from type I, type II, or type III CRISPR systems. In some embodiments, one or more elements of the CRISPR system are derived from specific organisms that include endogenous CRISPR systems, such as Streptococcus pyogenes.

[0172] Generally, the CRISPR system is characterized by elements that facilitate the formation of a CRISPR complex at a site of a target sequence. In the context of CRISPR complex formation, the “target sequence” refers to a sequence in which the guide RNA is designed to be complementary, and hybridization between the target sequence and the guide RNA facilitates the formation of the CRISPR complex. Perfect complementarity is not necessarily required, as long as there is sufficient complementarity to induce hybridization and facilitate the formation of the CRISPR complex. The target sequence may include any polynucleotide, such as DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be located in an organelle of a eukaryotic cell, such as a mitochondria or chloroplast. In one aspect of the present invention, the recombination is homologous recombination.

[0173] Typically, in the context of an endogenous CRISPR system, the formation of a CRISPR complex (containing a guide RNA that hybridizes to a target sequence and complexes with one or more Cas proteins) results in a break of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs). While not wishing to be bound by theory, a tracr sequence that may or may consist of all or part of a wild-type tracr sequence (e.g., 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, for example, by hybridization with all or part of a tracr mate sequence operably linked to the guide RNA along at least part of the tracr sequence. In some embodiments, the tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of the CRISPR complex.

[0174] Similar to the target sequence, complete complementarity is not required if sufficient hybridization and / or binding is present to be functional. In some embodiments, when aligned, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence.

[0175] In some embodiments, one or more vectors driving the expression of one or more elements of the CRISPR system are introduced into a host cell such that the expression of the elements of the CRISPR system directs the formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide RNA linked to a tracr mate sequence, and a tracr sequence may each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements may be combined into a single vector, and one or more additional vectors may provide any components of the CRISPR system not included in the first vector. The CRISPR system elements combined in a single vector may be positioned in any preferred orientation, for example, one element located 5' ("upstream") or 3' ("downstream") relative to a second element. The coding sequence of one element may be located on the same or opposite strand of the coding "sequence" of the second element and oriented in the same or opposite direction. In some embodiments, a single promoter drives the expression of a transcript encoding the CriSPR enzyme and one or more guide RNAs, tracr mate sequences (optionally operably ligated to the guide sequence), and tracr sequences, which are embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in one intron). In some embodiments, the CRISPR enzyme, guide RNA, tracr mate sequence, and tracr sequence are operably ligated to the same promoter and expressed from the same promoter.

[0176] Non-exclusive examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs or modified versions. In some embodiments, an unmodified CRISPR enzyme, such as Cas9, has DNA cleavage activity. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at a location in the 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 approximately 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 the target sequence.

[0177] In some embodiments, the vector encodes a CRISPR enzyme that is mutated from the corresponding wild-type enzyme such that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, the substitution of aspartic acid to alanine in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes (S. pyogenes) converts Cas9 from a nuclease that cleaves both strands to a nickase (single-strand cleavage). Other examples of mutations that convert Cas9 to a nickase include, but are not limited to, H840A, N854A, and N863A. In embodiments of the present invention, nickase may be used for genome editing by homologous recombination.

[0178] Generally, the guide RNA is any polynucleotide sequence that hybridizes with the target sequence and has sufficient complementarity to the target polynucleotide sequence to direct the sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or greater, when optimally aligned using a suitable alignment algorithm.

[0179] In some embodiments, the guide RNA has a nucleotide length of approximately 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. In some embodiments, the guide sequence has a nucleotide length of approximately 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less. The ability of the guide sequence to direct sequence-specific binding of the CRISPR complex to the target sequence can be evaluated by any suitable assay. For example, sufficient components of a CRISPR system to form a CRISPR complex containing the guide RNA to be tested can be provided to host cells having the corresponding target sequence, for example, by transfection with a vector encoding components of the CRISPR sequence, and then preferential cleavage within the target sequence can be evaluated by, for example, the Surveyor assay described herein. Similarly, cleavage of a target polynucleotide sequence can be evaluated in vitro by providing a CRISPR complex comprising the target sequence, a guide RNA to be tested, and a control guide RNA different from the test guide RNA, and comparing the binding or cleavage rate at the target sequence between the test guide sequence reaction and the control guide sequence reaction. Other assays are also possible and will be conceivable to those skilled in the art. The guide RNA can be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the cellular genome.

[0180] The terms "gRNA" and "guide RNA" are interchangeable and refer to specific RNA sequences that recognize a target DNA or RNA region and direct an endonuclease to that location for gene editing.

[0181] gRNA typically consists of two parts: crisp RNA (crRNA), a 17-30 nucleotide sequence complementary to the target DNA, and tracr RNA, which acts as a binding scaffold for Cas nucleases. Any suitable engineered gRNA, or crRNA and tracrRNA, can be used as long as it is effective in recognizing the target DNA or RNA. The design of such gRNA, or crRNA and tracrRNA, is within the scope of the art of the art.

[0182] The present invention provides a CPN ("CPN-gRNA") containing a guide RNA for CRISPR-based systems as a functional domain. The CPN-gRNA according to the present invention is described herein.

[0183] The Cpn-gRNA according to the present invention can be used in CRISPR systems known to those skilled in the art. For example, suitable CRISPR systems include International Publication Nos. 2013 / 176772, 2013 / 188638, 2014 / 018423, 2014 / 022702, 2014 / 093661, 2014 / 093622, 2017 / 004279, 2017 / 189308, and 2018 / 119354. Examples include, but are not limited to, International Publication Nos. 2018 / 208998, International Publication Nos. 2019 / 089804, International Publication Nos. 2019 / 089910, International Publication Nos. 2019 / 089808, International Publication Nos. 2019 / 089796, International Publication Nos. 2019 / 089820, and International Publication Nos. 2021 / 087394, the contents of each of these publications are incorporated herein by reference in their entirety.

[0184] Functional domain oligonucleotides are antisense oligonucleotides that function as guide RNAs for CRISPR-based systems. The terms "sgRNA" and "guide RNA" are interchangeable and refer to specific RNA sequences that recognize a target DNA or RNA region of interest and direct an endonuclease to it for editing. A gRNA typically consists of two parts: a crispr RNA (crRNA), a 17-30 nucleotide sequence complementary to the target DNA, and a tracr RNA that acts as a binding scaffold for the Cas nuclease.

[0185] Any suitable engineered sgRNA, crRNA, or tracrRNA can be used, as long as it is effective in recognizing the target DNA or RNA. The design of such sgRNA, crRNA, or tracrRNA is within the scope of the art of the art.

[0186] In embodiments where the functional domain of the CPN is an antisense oligonucleotide for a CRISPR-based system, the lengths of the first and second nucleic acid molecules can be increased. For example, the first and second nucleic acid molecules each independently contain an oligonucleotide with a length of 6 to 50 nucleotides. In some embodiments, the first and second nucleic acid molecules of the cyclic domain each independently contain an oligonucleotide with a length of 6 to 25 nucleotides. In some embodiments, the first and second nucleic acid molecules each independently contain an oligonucleotide with a length of 6 to 12 nucleotides. In some embodiments, the first and second nucleic acid molecules each independently contain an oligonucleotide with a length of 6 to 10 nucleotides. In some embodiments, the first and second nucleic acid molecules each independently contain an oligonucleotide with a length of 6 to 8 nucleotides.

[0187] mRNA In various embodiments, the present invention provides cyclic prodrug nucleic acids (CPNs) comprising a functional domain including a nucleic acid molecule, specifically polynucleotides, primary constructs, and / or mRNAs that encode one or more target polypeptides or fragments thereof and that possess sufficient structural and / or chemical characteristics to enable the encoding of the target polypeptides therein to be translated. Such target polypeptides may include, but are not limited to, whole polypeptides, plurality of polypeptides, or polypeptide fragments that can be independently encoded by one or more regions, parts, or the whole of a polynucleotide. As used herein, the term “target polypeptide” means any polypeptide selected to be encoded within the polynucleotide of the present invention, or whose function is influenced by the polynucleotide of the present invention.

[0188] In preferred embodiments, the nucleic acid molecule is messenger RNA (mRNA). As used herein, the term “messenger RNA” (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded polypeptide of interest.

[0189] In various 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.

[0190] In various embodiments, the mRNA molecule includes one or more structural and / or chemical modifications or alterations that confer useful properties to the polynucleotide, including, in some embodiments, the absence of substantial induction of the innate immune response in the cell into which the polynucleotide has been introduced. As used herein, a “structural” feature or modification is one in which two or more linked nucleotides are inserted, deleted, replicated, inverted, or randomized within the polynucleotide without significant chemical alteration to the nucleotides themselves. Structural modifications result in different nucleotide sequences. For example, the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”. The same polynucleotide may be structurally modified from “ATCG” to “ATCCCG”, where the dinucleotide “CC” is inserted, resulting in a structural modification to the polynucleotide.

[0191] In various embodiments, the mRNA functional domain includes a first region of linked nucleotides flanked by a first flanking region and a second flaking region. This first region may, but is not limited, contain the target encoded polypeptide. The target polypeptide may contain one or more signal sequences encoded by a signal sequence region at its 5' end. The first flanking region may contain a region of linked nucleotides containing one or more complete or incomplete 5'UTR sequences. The first flanking region may also contain a 5' terminal cap. The second flanking region may contain a region of linked nucleotides containing one or more complete or incomplete 3'UTR sequences. The second flanking region may also contain a 3' tailing sequence.

[0192] In various embodiments, the first manipulation region bridges the 5' ends of the first region and the first flanking region. This manipulation region includes a start codon. Alternatively, the manipulation region may include any translation initiation sequence or signal containing a start codon.

[0193] In some embodiments, the second operating region bridges the 3' ends of the first region and the second flanking region. This second operating region includes a stop codon. Alternatively, the second operating region may include any translation initiation sequence or signal containing a stop codon. In some embodiments, multiple consecutive stop codons may be used.

[0194] In various embodiments, the shortest length of the first region of the primary construct of the present invention may be a nucleic acid sequence length sufficient to encode a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. In another embodiment, the length may be sufficient to encode a peptide of 2 to 30 amino acids, e.g., 5 to 30, 10 to 30, 2 to 25, 5 to 25, 10 to 25, or 10 to 20 amino acids. The length may be sufficient to encode a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25, or 30 amino acids, or a peptide of 40 amino acids or less, e.g., 35, 30, 25, 20, 17, 15, 14, 13, 12, 11, or 10 amino acids or less.

[0195] Generally, the length of the first region encoding the target polypeptide of the present invention is greater than about 30 nucleotides (for example, at least 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 more, or up to 100,000 nucleotides). As used herein, “first region” may be referred to as “coding region” or “coding region” or simply “first region”.

[0196] In some embodiments, mRNA has approximately 30 to approximately 100,000 nucleotides (for example, 30-50, 30-100, 30-250, 30-500, 30-1,000, 30-1,500, 30-3,000, 30-5,000, 30-7,000, 30-10,000, 30-25,000, 30-50,000, 30-70,000, 100-250, 100-500, 100-1,000, 100~1,500 pieces, 100~3,000 pieces, 100~5,000 pieces, 100~7,000 pieces, 100~10,000 pieces, 100~25,000 pieces, 100~50,000 pieces, 100~70,000 pieces, 100~100 ,000 pieces, 500~1,000 pieces, 500~1,500 pieces, 500~2,000 pieces, 500~3,000 pieces, 500~5,000 pieces, 500~7,000 pieces, 500~10,000 pieces, 500~25,000 pieces, 500 pieces ~50,000 pieces, 500~70,000 pieces, 500~100,000 pieces, 1,000~1,500 pieces, 1,000~2,000 pieces, 1,000~3,000 pieces, 1,000~5,000 pieces, 1,000~7,000 pieces, 1,000~10,000 pieces, 1,000~25,000 pieces, 1,000~50,000 pieces, 1,000~70,000 pieces, 1,000~100,000 pieces, 1,500~3,000 pieces, 1,500~5,000 pieces, 1, This includes 500-7,000 units, 1,500-10,000 units, 1,500-25,000 units, 1,500-50,000 units, 1,500-70,000 units, 1,500-100,000 units, 2,000-3,000 units, 2,000-5,000 units, 2,000-7,000 units, 2,000-10,000 units, 2,000-25,000 units, 2,000-50,000 units, 2,000-70,000 units, and 2,000-100,000 units.

[0197] In various embodiments, the first and second flanking regions may independently be in the range of 15 to 1,000 nucleotide lengths (e.g., exceeding 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).

[0198] In various embodiments, the tailing sequence may range from nonexistent 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). If the tailing region is a poly(A) tail, its length may be determined in units of poly(A) binding protein binding, or as a function thereof. In this embodiment, the poly(A) tail is long enough to bind to at least four monomers of the poly(A) binding protein. The poly(A) binding protein monomers bind to a stretch of approximately 38 nucleotides. Thus, poly(A) tails of approximately 80 and 160 nucleotides have been observed to be functional.

[0199] In some embodiments, the capping region may comprise a single cap or a series of nucleotides forming a cap. In this embodiment, the capping region may have a nucleotide length of 1 to 10, for example, 2 to 9, 3 to 8, 4 to 7, 1 to 5, 5 to 10, or at least 2, or 10 or less. In some embodiments, no cap is present.

[0200] In various embodiments, the first and second operating regions may be in the range of 3 to 40, for example 5 to 30, 10 to 20, 15, or at least 4, or 30 or less in nucleotide length, and may include one or more signal and / or restriction sequences in addition to start and / or stop codons.

[0201] In various embodiments, the mRNA functional domain may be designed to encode a polypeptide of interest selected from any of several target categories, including but not limited to, biologics, antibodies, vaccines, therapeutic proteins or peptides, cell-permeable peptides, secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletal proteins, intracellular membrane-binding proteins, nucleoproteins, proteins associated with human diseases, targeted moieties, or proteins encoded by the human genome that are useful in the field of research and discovery even though their therapeutic indications have not been identified. In various embodiments, the mRNA functional domain may encode a variant polypeptide having specific identity with a reference polypeptide sequence. As used herein, “reference polypeptide sequence” means a starting polypeptide sequence. The reference sequence may be a wild-type sequence or any sequence referenced in the design of another sequence. The polypeptide variant may have the same or similar activity as the reference polypeptide. Alternatively, the variant may have modified (e.g., increased or decreased) activity compared to the reference polypeptide. Generally, when a particular variant of a polynucleotide or polypeptide of the present invention is determined by a sequence alignment program and parameters described herein and known to those skilled in the art, it has at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with that particular reference polynucleotide or polypeptide, but less than 100%.

[0202] In embodiments where the functional domain of the CPN is mRNA, the lengths of the first and second nucleic acid molecules can be longer compared to CPNs where the functional domain is a gene-regulating oligonucleotide. For example, the first and second nucleic acid molecules each independently contain an oligonucleotide of 6 to 50 nucleotides in length. In some embodiments, the first and second nucleic acid molecules of the cyclic domain each independently contain an oligonucleotide of 6 to 25 nucleotides in length. In some embodiments, the first and second nucleic acid molecules each independently contain an oligonucleotide of 6 to 12 nucleotides in length. In some embodiments, the first and second nucleic acid molecules each independently contain an oligonucleotide of 6 to 10 nucleotides in length. In some embodiments, the first and second nucleic acid molecules each independently contain an oligonucleotide of 6 to 8 nucleotides in length.

[0203] Immunostimulatory oligonucleotides In various embodiments, the present invention provides cyclic prodrug nucleic acids (CPNs) comprising a functional domain containing an immunostimulant oligonucleotide.

[0204] In various embodiments, immunostimulatory oligonucleotides can induce an interferon response in vertebrate cells.

[0205] In some embodiments, the nucleotide sequence of the immunostimulatory oligonucleotide is not complementary to another nucleotide sequence, such as the target RNA, and does not bind to it. In this embodiment, the nucleotide sequence of the immunostimulatory oligonucleotide is not, for example, an antisense oligonucleotide and does not possess antisense activity.

[0206] In various embodiments, the immunostimulatory oligonucleotide is 11 to 40 nucleotides long. In various embodiments, the immunostimulatory oligonucleotide is 15 to 28 nucleotides long. In various embodiments, the immunostimulatory oligonucleotide is 17 to 25 nucleotides long.

[0207] In various embodiments, the internucleotide bonds of immunostimulatory oligonucleotides are phosphorothioates, phosphodiesters, or combinations thereof.

[0208] In various embodiments, the nucleotide-nucleotide bonds of immunostimulatory oligonucleotides are phosphorothioate nucleotide bonds.

[0209] In various embodiments, the internucleotide bonds of immunostimulatory oligonucleotides are phosphodiesters.

[0210] In various embodiments, the internucleotide bonds of immunostimulatory oligonucleotides are a combination of nucleotide bonds of phosphorothioates and phosphodiesters.

[0211] Immunostimulatory oligonucleotides include, but are not limited to, oligonucleotides that are pathogen-associated molecular patterns (PAMPS) for pattern recognition receptors (PRRs).

[0212] Immunostimulatory oligonucleotides include, but are not limited to, oligonucleotides that induce immunostimulation via endosomal Toll-like receptors, RIG-like receptors, STING, cGAS, and inflammasomes. Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that play a crucial role in initiating the innate immune response by detecting potential harmful pathogens. Each TLR has a broad specificity; for example, TLR1, 2, 4, and 6 recognize bacterial lipids, TLR3, 7, and 8 recognize viral RNA, TLR9 recognizes bacterial DNA containing CG motifs, and TLR5 and 10 recognize bacterial or parasitic proteins. The design of such immunostimulatory oligonucleotides is within the scope of the art of the art.

[0213] Immunoant antagonist In various embodiments, the present invention provides cyclic prodrug nucleic acids (CPNs) comprising a functional domain containing an immunoantagonist oligonucleotide.

[0214] In various embodiments, immune antagonist oligonucleotides can block the interferon response in vertebrate cells.

[0215] In some embodiments, the nucleotide sequence of the immune antagonist oligonucleotide is not complementary to another nucleotide sequence, such as the target RNA, and does not bind to it. In this embodiment, the nucleotide sequence of the immune antagonist oligonucleotide is not, for example, an antisense oligonucleotide and does not have antisense activity.

[0216] In various embodiments, the immunoantagonist oligonucleotide is 11 to 30 nucleotides long. In various embodiments, the immunoantagonist oligonucleotide is 15 to 28 nucleotides long. In various embodiments, the immunoantagonist oligonucleotide is 17 to 25 nucleotides long.

[0217] In various embodiments, the internucleotide bonds of the immunoantagonist oligonucleotides are phosphorothioates, phosphodiesters, or combinations thereof.

[0218] In various embodiments, the nucleotide-nucleotide bonds of the immunoantagonist oligonucleotides are phosphorothioate nucleotide bonds.

[0219] In various embodiments, the internucleotide bonds of the immunoantagonist oligonucleotides are phosphodiesters.

[0220] In various embodiments, the internucleotide bonds of the immunoantagonist oligonucleotide are a combination of nucleotide bonds of phosphorothioates and phosphodiesters.

[0221] Immune antagonist oligonucleotides include, but are not limited to, oligonucleotides that block immune stimulation via endosomal toll-like receptors, RIG-like receptors, STING, cGAS, and inflammasomes. Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that play an important role in initiating the innate immune response by detecting potential harmful pathogens. Each TLR has a wide range of specificities. For example, TLR1, 2, 4, and 6 recognize bacterial lipids, TLR3, 7, and 8 recognize viral RNA, TLR9 recognizes bacterial DNA containing CG motifs, and TLR5 and 10 recognize bacterial or parasite proteins. The design of such immune antagonist oligonucleotides is within the scope of the skill of those skilled in the art.

[0222] siRNA In some embodiments, the oligonucleotide of the functional domain is siRNA. siRNA includes short double-stranded RNAs that target RNA and are about 15 to about 50 nucleotides in length, preferably about 18 to about 36 nucleotides in length.

[0223] In some embodiments, the present invention provides a CPN comprising a functional domain and a cyclization domain, the functional domain comprising siRNA; the cyclization domain comprising a first nucleic acid molecule and a second nucleic acid molecule, the first nucleic acid molecule and the second nucleic acid molecule being linked to the siRNA of the functional domain directly or via a linker segment; the first nucleic acid molecule and the second nucleic acid molecule being independently 6 to 30 nucleotides in length, the nucleotides of the first nucleic acid molecule and the second nucleic acid molecule being independently selected from RNA or DNA or a combination of RNA and DNA, the first nucleic acid molecule and the second nucleic acid molecule being complementary to each other and of opposite polarities and hybridizing to form a double-stranded portion.

[0224] The terms "siRNA" and "small interfering RNA" are interchangeable and refer to single-stranded or double-stranded RNA molecules capable of inducing RNA interference. siRNA molecules typically have a double-stranded region that is 18 to 36 base pairs in length. The design of such siRNAs is within the skill of those in the art.

[0225] In one embodiment, when the oligonucleotide of the functional domain is siRNA, the first nucleic acid molecule of the cyclization domain binds to the 5' end of the sense strand of the siRNA, and the second nucleic acid molecule of the cyclization domain binds to the 3' end of the sense strand of the siRNA.

[0226] In one embodiment, when the oligonucleotide of the functional domain is siRNA, the first nucleic acid molecule of the cyclization domain binds to the 5' end of the antisense strand of the siRNA, and the second nucleic acid molecule of the cyclization domain binds to the 3' end of the antisense strand of the siRNA.

[0227] In one embodiment, when the oligonucleotide of the functional domain is siRNA, the first nucleic acid molecule of the cyclization domain binds to the 5' end of the antisense strand of the siRNA, and the second nucleic acid molecule of the cyclization domain binds to the 5' end of the sense strand of the siRNA.

[0228] In one embodiment, when the oligonucleotide of the functional domain is siRNA, the first nucleic acid molecule of the cyclization domain binds to the 3' end of the antisense strand of the siRNA, and the second nucleic acid molecule of the cyclization domain binds to the 3' end of the sense strand of the siRNA.

[0229] One or both strands of the siRNA of the present invention may also include a 3'-overhang. "3' overhang" refers to at least one unpaired nucleotide extending from the 3' end of an RNA strand. Thus, in one embodiment, the siRNA of the present invention includes at least one 3'-overhang that is 1 to about 6 nucleotides in length (including ribonucleotides or deoxynucleotides), preferably 1 to about 5 nucleotides in length, more preferably 1 to about 4 nucleotides in length, and particularly preferably about 1 to about 2 nucleotides in length.

[0230] If both strands of the siRNA molecule contain a 3' overhang, the length of the overhang may be the same or different for each strand. In the most preferred embodiment, the 3' overhang is present on both strands of the siRNA and is 2 nucleotides long. To enhance the stability of the siRNA, the 3' overhang can also be stabilized against degradation. In one embodiment, the overhang is stabilized by including a purine nucleotide, such as adenosine or guanosine nucleotide.

[0231] Alternatively, substitution of pyrimidine nucleotides with modified analogs, such as substitution of uridine nucleotides in the 3' overhang with 2'-deoxythymidine, is acceptable and does not affect the efficiency of RNAi degradation. In particular, the absence of a 2' hydroxyl group in 2'-deoxythymidine significantly enhances nuclease resistance of the 3' overhang in tissue culture medium.

[0232] The siRNA of the present invention can target any stretch of approximately 18 to 30, preferably 19 to 25, consecutive nucleotides in the target mRNA sequence. Techniques for selecting target sequences for siRNA are well known in the art. Therefore, the sense strand of this siRNA contains the same nucleotide sequence as any stretch of approximately 18 to approximately 30 consecutive nucleotides in the target mRNA.

[0233] Pharmaceutical composition In certain embodiments, the pharmaceutical composition described herein comprises one or more CPN compounds of the present invention. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier. In certain embodiments, the pharmaceutical composition comprises sterile saline and one or more CPN compounds. In certain embodiments, the pharmaceutical composition consists of sterile saline and one or more CPN compounds. In certain embodiments, the sterile saline is pharmaceutical-grade saline. In certain embodiments, the pharmaceutical composition comprises one or more CPN compounds and sterile water. In certain embodiments, the pharmaceutical composition consists of one CPN compound and sterile water. In certain embodiments, the sterile water is pharmaceutical-grade water. In certain embodiments, the pharmaceutical composition comprises one or more CPN compounds and phosphate-buffered saline (PBS). In certain embodiments, the pharmaceutical composition consists of one or more CPN compounds and sterile PBS. In certain embodiments, the sterile PBS is pharmaceutical-grade PBS.

[0234] In certain embodiments, the pharmaceutical composition comprises one or more CPN compounds and one or more excipients. In certain embodiments, the excipients are selected from water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0235] In certain embodiments, the CPN compounds of the present invention may be mixed with pharmaceutically acceptable active and / or inactive substances for the preparation of pharmaceutical compositions or formulations. The compositions and methods for formulating pharmaceutical compositions are not limited but depend on many criteria, including the route of administration, the severity of the disease, or the dose administered.

[0236] conjugate group In certain embodiments, the CPN according to the present invention optionally further comprises one or more conjugate groups. The conjugate group consists of one or more conjugate moieties and a conjugate linker that links one or more conjugate moieties to an oligonucleotide. The conjugate group may be bonded to one or both ends and / or any internal position of the oligonucleotide. In certain embodiments, the conjugate group is bonded to the 2' position of the nucleoside of the modified oligonucleotide. In certain embodiments, the conjugate group bonded to one or both ends of the oligonucleotide is a terminal group. In certain such embodiments, the conjugate group or terminal group is bonded to the 3' and / or 5' ends of the oligonucleotide. In certain such embodiments, the conjugate group is bonded to the 3' end of the oligonucleotide. In certain embodiments, the conjugate group is bonded near the 3' end of the oligonucleotide. In certain embodiments, the conjugate group is bonded to the 5' end of the oligonucleotide. In certain embodiments, the conjugate group is bonded near the 5' end of the oligonucleotide.

[0237] In any embodiment of this specification, the conjugate group comprises a GalNAc cluster containing 1 to 3 GalNAc ligands.

[0238] In any embodiment described herein, the conjugate linker consists of a single bond.

[0239] In any embodiment described herein, the conjugate linker is detachable.

[0240] In any embodiment described herein, the conjugate linker comprises 1 to 3 linker-nucleosides.

[0241] In any embodiment of this specification, the conjugate group is bound to the CPN at the end of a first nucleic acid molecule and / or a second nucleic acid molecule. The end of the first or second nucleic acid molecule is the end of the CPN of the present invention that is not bound to an oligonucleotide.

[0242] In certain embodiments, the CPN is covalently bonded to one or more conjugate groups. In certain embodiments, the conjugate groups modify one or more properties of the CPN, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cell distribution, cell uptake, charge, and clearance. In certain embodiments, the conjugate groups confer new properties to the CPN, such as a fluorophore or reporter group that enables the detection of oligonucleotides. Specific conjugate groups and conjugate moieties include, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NYA Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), and thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992,20,533-538), aliphatic chains, e.g., dodecane-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), phospholipids, e.g., dihexadecyl-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), polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969 - 973), or an adamantane acetic acid palmitoyl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229 - 237), an octadecylamine or hexylamino-carbonyl-oxy cholesterol 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., International Publication No. 2014 / 179620) has been previously described.

[0243] The conjugate moiety includes, but is not limited to, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycol, thioethers, polyethers, cholesterol, thiocolesterol, cholanic acid moieties, folic acid, lipids, phospholipids, biotin, phenazines, phenanthridines, anthraquinones, adamantanes, acridines, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.

[0244] In certain embodiments, the conjugate moiety includes an active pharmaceutical substance, such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folic acid, benzothiadiazide, chlorothiazide, diazepine, indomethacin, barbiturate, cephalosporin, sulfonamide, antidiabetic drug, antibacterial drug, or antibiotic.

[0245] The conjugate portion is bonded to the CPN via a conjugate linker. In certain embodiments, the conjugate linker is a single chemical bond (i.e., the conjugate portion is directly bonded to the CPN via a single bond). In certain embodiments, the conjugate linker comprises a chain structure such as a hydrocarbyl chain, or an oligomer of repeating units such as ethylene glycol, a nucleoside, or an amino acid unit.

[0246] In certain embodiments, the conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises a group selected from alkyl, amino, oxo, amide, and ether groups. In certain embodiments, the conjugate linker comprises a group selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises a group selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus moiety. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker comprises at least one neutral linking group.

[0247] In certain embodiments, conjugate linkers, including the above-described conjugate linker, are known in the art to be useful for attaching a conjugate group to a parent compound, such as a difunctional linkage, for example, an oligonucleotide provided herein. Generally, a difunctional linkage includes at least two functional groups. One of the functional groups is selected to bond to a specific site on the CPN, and the other is selected to bond to a conjugate group. Examples of functional groups used in a difunctional linkage include, but are not limited to, electrophiles for reacting with nucleophiles and nucleophiles for reacting with electrophiles. In certain embodiments, the difunctional linkage includes one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl groups.

[0248] 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-C 10 Alkyl, substituted, or unsubstituted C2-C 10 Alkenyl or substituted or unsubstituted C2-C 10 Examples of alkynyl substituents include hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl substituents.

[0249] In certain embodiments, the conjugate linker contains 1 to 10 linker-nucleosides. In certain embodiments, the conjugate linker contains 2 to 5 linker-nucleosides. In certain embodiments, the conjugate linker contains exactly 3 linker-nucleosides. In certain embodiments, the conjugate linker contains a TCA motif. In certain embodiments, such linker-nucleosides are modified nucleosides. In certain embodiments, such linker-nucleosides contain a modified sugar moiety. In certain embodiments, the linker-nucleosides are unmodified. In certain embodiments, the linker-nucleosides contain an optionally protected heterocyclic base selected from purines, substituted purines, pyrimidines, or substituted pyrimidines. In certain embodiments, the cleavable portion is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine, and 2-N-isobutyrylguanine. It is typically desirable that the linker-nucleoside be cleaved from the oligomer compound after reaching the target tissue. Thus, the linker-nucleosides are typically linked to each other and to the rest of the oligomer compound via cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.

[0250] As used herein, linker nucleosides are not generally considered part of the CPN, or in particular part of the cyclic domain or functional domain. Therefore, the nucleotides of linker nucleosides are not counted against the length of the CPN or its domain and are not used to determine the complementarity percentage of oligonucleotides to the reference nucleic acid. Unless otherwise indicated, a conjugate linker contains 10 or fewer linker nucleosides. In certain embodiments, a conjugate linker contains 5 or fewer linker nucleosides. In certain embodiments, a conjugate linker contains 3 or fewer linker nucleosides. In certain embodiments, a conjugate linker contains 2 or fewer linker nucleosides. In certain embodiments, a conjugate linker contains 1 or fewer linker nucleosides.

[0251] In certain embodiments, it is desirable that the conjugate group be cleaved from the CPN. For example, in certain circumstances, a CPN containing a particular conjugate moiety is better taken up by a particular cell type, but it is desirable that once the CPN is taken up, the conjugate group is cleaved to release the unconjugated or parent CPN. Therefore, a particular conjugate linker may contain 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 containing at least one cleavable bond. In certain embodiments, a cleavable moiety includes a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, a cleavable moiety is selectively cleaved within a cell or an intracellular compartment such as a lysosome. In certain embodiments, a cleavable moiety is selectively cleaved by an endogenous enzyme such as a nuclease.

[0252] In certain embodiments, the cleavable bond is selected from amides, esters, ethers, one or both phosphodiesters, phosphate esters, carbamates, or disulfides. In certain embodiments, the cleavable bond is one or both phosphodiesters. In certain embodiments, the cleavable portion includes phosphate or a phosphodiester. In certain embodiments, the cleavable portion is a phosphate bond between the oligonucleotide and the conjugate moiety or conjugate group.

[0253] use The cyclic prodrug oligonucleotides of the present invention may be useful for a variety of mechanisms of action. These include mRNA, ncRNA, microRNA, lncRNA, and antisense for splicing. Furthermore, the double-stranded cyclic prodrug structure can be used to deliver siRNA constructs. In addition, the cyclic prodrug structure can provide a novel approach to delivering antisense for ADAR and CRISPR-based mechanisms. Moreover, the cyclic prodrug structure can provide a novel approach to delivering antisense to disrupt RNA structure and increase translation. The cyclic prodrug structure also provides a defined structure for nucleic acids, thereby enabling varying degrees of interaction with PRRs and induced immune cascades.

[0254] For example, if the functional domain is an antisense oligonucleotide, the CPN according to the present invention is also useful in therapeutic approaches where inhibition of gene expression is desired. This may include, for example, inhibition of endogenous genes (e.g., oncogenes) or exogenous genes (e.g., genes essential for pathogen growth and / or metabolism).

[0255] In various embodiments, the present invention provides a method for inhibiting gene expression, comprising administering a cyclic prodrug nucleic acid or a composition comprising a cyclic prodrug nucleic acid as described herein.

[0256] In various embodiments, the present invention provides a method for inhibiting allele-specific gene expression, comprising administering a cyclic prodrug nucleic acid or a composition comprising a cyclic prodrug nucleic acid as described herein.

[0257] The methods according to the present invention are useful for treating subjects with diseases or disorders in which inhibiting gene expression is beneficial. In various embodiments, the disease or disorder is caused by abnormal expression or products of cellular genes.

[0258] In various embodiments, the cyclic prodrug nucleic acid or composition comprising the cyclic prodrug nucleic acid described herein is administered topically.

[0259] In various embodiments, the cyclic prodrug nucleic acid or composition comprising the cyclic prodrug nucleic acid described herein is administered systemically.

[0260] In some embodiments, a method for modulating RNA processing comprises administering a CPN compound described herein, wherein the functional domain comprises an antisense oligonucleotide comprising 14 to 30 linked nucleotides having at least 12 consecutive nucleic acid bases complementary to equal-length portions of the target RNA, the antisense oligonucleotide comprising 1 to 3 DNA regions, each region independently comprising 2 to 5 consecutive deoxyribonucleotides, and the remaining nucleotides being 2'-substituted, nonionic, or constrained sugar nucleotides, or a combination thereof. In some embodiments, RNA processing comprises splicing.

[0261] In various embodiments, the present invention provides a method for selecting a first mRNA transcript in a gene containing at least two mRNA transcripts, comprising administering a cyclic prodrug nucleic acid or a composition containing a cyclic prodrug nucleic acid as described herein.

[0262] In various embodiments, the present invention provides a method for treating a disease or disorder of interest, wherein modulating RNA processing is beneficial for treating the subject, and the method comprises administering a cyclic prodrug nucleic acid or a composition comprising a cyclic prodrug nucleic acid as described herein.

[0263] In various embodiments, the present invention provides a method for inducing a nonsense mutation-dependent mRNA degradation mechanism of a target RNA, comprising administering a cyclic prodrug nucleic acid or a composition comprising a cyclic prodrug nucleic acid as described herein.

[0264] In various embodiments, the present invention provides a method for increasing the level of mRNA encoding a protein or functional mRNA and increasing the expression of a protein or functional mRNA, comprising administering a cyclic prodrug nucleic acid or a composition comprising a cyclic prodrug nucleic acid as described herein.

[0265] The cyclic prodrug nucleic acids of the present invention may be administered alone or in combination with any other agent or treatment. The agents or treatments may be co-administered or administered simultaneously. Such agents or treatments may be useful for treating or preventing diseases or symptoms and will not diminish the gene expression regulatory effect of the cyclic prodrug nucleic acids according to the present invention. Agents useful for treating or preventing diseases or symptoms include, but are not limited to, small molecules, peptide vaccines, antigens, antibodies, preferably monoclonal antibodies, cytotoxic agents, kinase inhibitors, allergens, antibiotics, siRNA molecules, antisense oligonucleotides, TLR antagonists (e.g., TLR3 and / or TLR7 antagonists and / or TLR8 antagonists and / or TLR9 antagonists), chemotherapeutic agents (both traditional chemotherapy and modern targeted therapies), targeted therapeutic agents, activated cells, peptides, proteins, gene therapy vectors, peptide vaccines, protein vaccines, DNA vaccines, adjuvants, and costimulatory molecules (e.g., cytokines, chemokines, protein ligands, transactivators, peptides containing peptides or modified amino acids), or combinations thereof. Alternatively, the cyclic prodrug nucleic acid according to the present invention may be administered in combination with other compounds (e.g., lipids or liposomes) to enhance the specificity or magnitude of gene expression regulation of the cyclic prodrug nucleic acid according to the present invention.

[0266] The cyclic prodrug nucleic acid of the present invention may be administered by any suitable route, including but not limited to parenteral, mucosal delivery, oral, sublingual, transdermal, topical, inhalation, intratumoral, intravenous, subcutaneous, intrathecal, intranasal, aerosol, intraocular, intratracheal, rectal, vaginal, gene gun, skin patch, or in the form of eye drops or mouthwash. In any of the methods according to the present invention, the cyclic prodrug nucleic acid of the present invention may be administered directly to a tissue or organ, such as but not limited to the bladder, liver, lungs, or kidneys, either alone or in combination with any other agent. In certain embodiments, the administration of the cyclic prodrug nucleic acid of the present invention, either alone or in combination with any other agent, is by intramuscular administration. In certain embodiments, the administration of the cyclic prodrug nucleic acid of the present invention, either alone or in combination with any other agent, is by mucosal administration. In certain embodiments, the administration of the cyclic prodrug nucleic acid of the present invention, either alone or in combination with any other agent, is by oral administration. In certain embodiments, the administration of the cyclic prodrug nucleic acid of the present invention, either alone or in combination with any other agent, is by rectal administration. In certain embodiments, administration of the cyclic prodrug nucleic acid according to the present invention, either alone or in combination with any other agent, is by intrathecal administration. In certain embodiments, administration of the cyclic prodrug nucleic acid according to the present invention, either alone or in combination with any other agent, is by intratumoral administration. In certain embodiments, administration of the cyclic prodrug nucleic acid according to the present invention, either alone or in combination with any other agent, is by parenteral administration. In certain embodiments, administration of the cyclic prodrug nucleic acid according to the present invention, either alone or in combination with any other agent, is by subcutaneous administration.

[0267] In various embodiments, any of the cyclic prodrug nucleic acids described herein can be conjugated with a moiety that provides site-specific delivery of the CPN. In various embodiments, such conjugates include, but are not limited to, antibodies, peptides, lipids, or small molecules.

[0268] In various embodiments, any of the cyclic prodrug nucleic acids described herein can be encapsulated in a portion that provides site-specific delivery of the CPN. In various embodiments, the CPN can be encapsulated in, for example, lipids, lipid nanoparticles (LNPs), or macrocyclic peptide structures.

[0269] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents, e.g., water for injection, saline, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetic acid, citric acid, or phosphoric acid; and agents for adjusting isotonicity such as sodium chloride or dextrose. pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials. Administration of the cyclic prodrug nucleic acid according to the present invention may be carried out using known procedures in an effective amount for a period effective in reducing the symptoms or surrogate markers of the disease. For example, an effective amount of the cyclic prodrug nucleic acid according to the present invention for treating a disease and / or disorder may be the amount necessary to alleviate or reduce symptoms or to delay or induce remission of tumors, cancer, or bacterial, viral, or fungal infections. In the context of administering compositions that modulate gene expression, the effective amount of the cyclic prodrug nucleic acid according to the present invention is sufficient to achieve the desired modulation compared to gene expression in the absence of the antisense oligonucleotide according to the present invention. The effective amount for any particular application may vary depending on factors such as the disease or symptom being treated, the specific oligonucleotide administered, the size of the subject, or the severity of the disease or symptom. Those skilled in the art can empirically determine the effective amount of a particular cyclic prodrug nucleic acid without requiring excessive experimentation.

[0270] synthesis The CPNs described herein can be prepared by any suitable method recognized in the art, including but not limited to H-phosphonate chemistry, phosphoramidite chemistry, or a combination of H-phosphonate chemistry and phosphoramidite chemistry (i.e., H-phosphonate chemistry in some cycles and phosphoramidite chemistry in other cycles), which can be done manually or by an automated synthesizer. The oligonucleotides of the present invention can also be modified in several ways without impairing their ability to hybridize to their targets (see, for example, Agrawal and Gait, Advances in Nucleic Acid Therapeutics, (2019) https: / / doi.org / 10.1039 / 9781788015714).

[0271] definition Unless otherwise specified, the nomenclature, procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are well known and commonly used in the art. Where permitted, all patents, applications, published applications, and other publications and other data referenced throughout this disclosure are incorporated herein by reference in their entirety.

[0272] Unless otherwise specified, the following terms have the following meanings: As used herein, “2'-deoxynucleoside” means a nucleoside containing a 2'-H(H) furanosyl sugar moiety found in naturally occurring deoxyribonucleic acid (DNA). In certain embodiments, the 2'-deoxynucleoside may contain a modified nucleic acid base or an RNA nucleic acid base (uracil).

[0273] As used herein, “2'-substituted nucleoside” means a nucleoside containing a 2'-substituted sugar moiety. As used herein, with respect to the sugar moiety, “2'-substituted” means a sugar moiety containing at least one 2'-substituent other than H or OH.

[0274] As used herein, "5-methylcytosine" means cytosine modified by a methyl group attached to the 5-position. 5-methylcytosine is a modified nucleic acid base.

[0275] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless otherwise explicitly indicated by the context.

[0276] As used herein, “administer” means to provide a medicinal product to an animal.

[0277] As used herein, “animal” means human or non-human animal.

[0278] As used herein, “individuals requiring it” refers to humans or non-human animals selected for a procedure or treatment that requires such a procedure or treatment.

[0279] As used herein, “antisense activity” means any detectable and / or measurable change resulting from the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of the target nucleic acid or the protein encoded by such target nucleic acid compared to the level of the target nucleic acid or the level of the target protein in the absence of the antisense compound. In certain embodiments, antisense activity is an increase in the amount or expression of the target nucleic acid or the protein encoded by such target nucleic acid compared to the level of the target nucleic acid or the level of the target protein in the absence of the antisense compound.

[0280] As used herein, “antisense compound” means an oligomeric compound capable of achieving at least one antisense activity.

[0281] As used herein, “to induce remission” with respect to a treatment means improvement of at least one symptom compared to the same symptom in the absence of the treatment. In certain embodiments, remission is a reduction in the severity or frequency of a symptom, or a delay in the onset or progression of the severity or frequency of a symptom. In certain embodiments, the symptoms or features are ataxia, neuropathy, and aggregate formation. In certain embodiments, remission of these symptoms results in improvement of motor function, reduction of neuropathy, or reduction in the number of aggregates.

[0282] As used herein, “bicyclic nucleoside” or “BNA” means a nucleoside containing a bicyclic sugar moiety. As used herein, “bicyclic sugar” or “bicyclic sugar moiety” means a modified sugar moiety containing two rings, the second ring being formed via a bridge connecting two atoms of the first ring, thereby forming a bicyclic structure. In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In certain embodiments, the bicyclic sugar moiety does not contain a furanosyl moiety.

[0283] As used herein, “chiral-enriched population” means multiple molecules of the same molecular formula, where the number or percentage of molecules in the population containing 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 in the population if the particular chiral center is sterically random. A chiral-enriched population of molecules having multiple chiral centers within each molecule may contain one or more sterically random chiral centers. In certain embodiments, the molecule is a modified oligonucleotide. In certain embodiments, the molecule is a compound comprising a modified oligonucleotide.

[0284] As used herein, “cleavable portion” means a group of bonds or atoms that are cleaved under physiological conditions, for example, within a cell, animal, or human.

[0285] As used herein, the term “complementary” refers to a pair of nucleic acid bases (or simply “bases”) that, under selected (e.g., physiological) conditions, bond hydrogen to each other more favorably than other heterocyclic bases (or the degree of complementarity that may be required in context when evaluating the “complementarity” of an oligonucleotide). When the nucleic acid bases are modified or unmodified, natural or synthetic purines and pyrimidines, the term “complementary” means complementary in the Watson-Crick sense. In some embodiments, when the nucleic acid base sequence of an oligonucleotide is aligned in opposite directions to the nucleic acid sequence of another nucleic acid, at least 70% of the nucleic acid bases of the oligonucleotide or one or more regions thereof and the nucleic acid bases of the other nucleic acid or one or more regions thereof can bond hydrogen to each other. Complementary nucleic acid bases refer to nucleic acid bases that can form hydrogen bonds with each other.

[0286] Complementary nucleic acid base pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine (mC) and guanine (G). Complementary oligonucleotides and / or nucleic acids do not need to have nucleic acid base complementarity at each nucleoside. Rather, some mismatch is acceptable. As used herein, "perfectly complementary" or "100% complementary" with respect to an oligonucleotide means that the oligonucleotide is complementary to another oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide.

[0287] As used herein, “conjugate group” means a group of atoms that are directly or indirectly bonded to an oligonucleotide. A conjugate group comprises a conjugate moiety and a conjugate linker that bonds the conjugate moiety to an oligonucleotide.

[0288] As used herein, “conjugate linker” means a group of atoms containing at least one bond that connects the conjugate portion to an oligonucleotide.

[0289] As used herein, “conjugate moiety” means the group of atoms bonded to an oligonucleotide via a conjugate linker.

[0290] As used herein, “sequential” in the context of oligonucleotides refers to nucleosides, nucleic acid bases, sugar moieties, or internucleoside bonds that are immediately adjacent to each other. For example, “sequential nucleic acid bases” means nucleic acid bases that are immediately adjacent to each other in sequence.

[0291] As used herein, "linker-nucleoside" means a nucleoside that directly or indirectly links the CPN of the present invention to the conjugate portion. The linker-nucleoside is located within the conjugate linker and, even if continuous with the CPN, is not considered part of the CPN compound.

[0292] As used herein, “gapmer” means a modified oligonucleotide comprising an internal region having multiple nucleosides supporting RNase H cleavage located between an external region having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleosides comprising the external region (one or more). The internal region may be called a “gap,” and the external region may be called a “wing.” Unless otherwise indicated, “gapmer” refers to a sugar motif. Unless otherwise indicated, the sugar portion of the nucleoside in the gap of a gapmer is unmodified 2'-deoxyfuranosyl. Thus, the term “MOE gapmer” refers to a gapmer having a sugar motif of 2'-MOE nucleoside and a gap of 2'-deoxynucleoside in both wings. Unless otherwise indicated, a MOE gapmer may comprise one or more modified internucleoside bonds and / or modified nucleic acid bases, and such modifications do not necessarily follow the gapmer pattern of sugar modifications.

[0293] As used herein, “hotspot region” is a range of nucleic acid bases on a target nucleic acid suitable for an oligomeric compound to reduce the amount or activity of the target nucleic acid, as demonstrated in the following examples herein.

[0294] As used herein, “hybridization” means the pairing or annealing of complementary oligonucleotides and / or nucleic acids. While not limited to specific mechanisms, the most common mechanisms of hybridization involve hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds between complementary nucleic acid bases.

[0295] As used herein, the term “nucleoside bond” refers to a covalent bond between adjacent nucleosides in an oligonucleotide. As used herein, “modified nucleoside bond” refers to any nucleoside bond other than a phosphodiester nucleoside bond. A “phosphothioate bond” is a modified nucleoside bond in which one of the non-bridged oxygen atoms of a phosphodiester nucleoside bond is replaced with a sulfur atom.

[0296] As used herein, the phrase "inhibit expression or activity" refers to a reduction or blockage of expression or activity compared to the expression or activity in the untreated or control sample, and does not necessarily mean complete elimination of expression or activity.

[0297] As used herein, “non-bicyclic modified sugar moiety” means a modified sugar moiety that does not form a bridge between two atoms of the sugar to form a second ring, for example, a modified sugar moiety that includes substituents.

[0298] As used herein, “mismatch” or “non-complementary” means that, when the first and second oligomer compounds are aligned, the nucleic acid base of the first oligonucleotide is not complementary to the corresponding nucleic acid base of the second oligonucleotide or the target nucleic acid.

[0299] As used herein, "MOE" means methoxyethyl. "2'-MOE" means a 2'-OCH2CH2OCH3 group instead of a 2'OH group in the ribosyl sugar moiety.

[0300] As used herein, “motif” means the pattern of unmodified and / or modified sugar moieties, nucleic acid bases, and / or nucleoside bonds in an oligonucleotide.

[0301] As used herein, "mRNA" means a protein-coding RNA transcript, and unless otherwise specified, includes pre-mRNA and mature mRNA.

[0302] As used herein, “nucleic acid base” means either an unmodified or modified nucleic acid base. As used herein, “unmodified nucleic acid base” refers to adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G). As used herein, “modified nucleic acid base” refers to a group of atoms other than unmodified A, T, C, U, or G that can pair with at least one unmodified nucleic acid base. “5-methylcytosine” is an example of a modified nucleic acid base. A universal base is a modified nucleic acid base that can pair with any one of the five unmodified nucleic acid bases. Modified bases, also called heterocyclic base moieties, include other nucleic acid bases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, and alkynyl derivatives of other pyrimidine bases, 6-azouracil, and cytosine. This also includes thymine, 5-uracil (pseudracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (including 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosine), 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0303] In certain embodiments, the modified nucleic acid bases are selected from the universal bases, hydrophobic bases, indiscriminate bases, size-expanded bases, and fluorinated bases as defined herein. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines (including 2-aminopropyladenine and 5-propynyluracil); 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, and alkynyl derivatives of other pyrimidine bases, and 6-azoura. Cyl, cytosine and thymine, 5-uracil (pseudracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine. Further modified nucleic acid bases include tricyclic pyrimidines, such as phenoxazinecytidine ([5,4-b][1,4]benzoxazine-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiadin-2(3H)-one), G-clamps, such as substituted phenoxazinecytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazine-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indole-2-one), and pyridoindolecytidine (H-pyrimido[3',2':4,5]pyrrolo[2,3-d]pyrimidine-2-one). Modified nucleic acid bases may also include those in which a purine base or pyrimidine base is substituted with another heterocycle, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. In certain embodiments, the modified nucleic acid base is 5-methylcytosine.

[0304] Typical modified sugars include carbocyclic or acyclic sugars, sugars having substituents on one or more of their 2', 3', or 4' positions, and sugars having substituents in place of one or more hydrogen atoms. In certain embodiments, sugars are modified by having a substituent at the 2' position. In additional embodiments, sugars are modified by having a substituent at the 3' position. In other embodiments, sugars are modified by having a substituent at the 4' position. Sugars may have two or more modifications at their positions, or antisense oligonucleotides may have one or more nucleotides with sugar modifications at one position, or one or more nucleotides with sugar modifications at different positions.

[0305] The sugar modifications intended in oligonucleotides include, but are not limited to, sugar substituents selected from the following: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl substituents are substituted or unsubstituted C1-C12. 10 Alkyl or C2-C 10 They can be alkenyls and alkynyls. In some embodiments, these groups are O(CH2) x OCH3, O((CH2) x O) y CH3, O(CH2) x NH2, O(CH2) x CH3, O(CH2) x ONH2 and O(CH2) x ON((CH2) x CH3)2 (where x and y are independently between 1 and 10) can be selected.

[0306] In some embodiments, the modified sugars are as follows: C1~C 10The substituents include lower alkyl groups, substituted lower alkyl groups, alkenyl groups, alkynyl groups, alkaryl groups, alkaryl groups, aralkyl groups, O-alkaryl groups or O-aralkyl groups, SH groups, SCH3 groups, Cl groups, Br groups, CN groups, OCN groups, CF3 groups, OCF3 groups, SOCH3 groups, SO2CH3 groups, ONO2 groups, NO2 groups, N3 groups, NH2 groups, heterocycloalkyl groups, heterocycloalkaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of antisense oligonucleotides, or groups for improving the pharmacodynamic properties of antisense oligonucleotides, and substituents selected from other substituents having similar properties. In one embodiment, the modification includes 2'-methoxyethoxy (2'-O-(2-methoxyethyl) or 2'-MOE, also known as 2'-O-CH2CH2OCH3) (Martin et al., 1995), i.e., an alkoxyalkoxy group. Another modification involves 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH 2- O-CH2-N(CH3)2 is one example.

[0307] Additional sugar substituents include allyl (-CH2-CH=CH2) and -O-allylCH 2-Examples include CH=CH2, methoxy(-O-CH3), aminopropoxy(-OCH2CH2CH2NH2), and fluoro(F). The sugar substituent at the 2' position (2'-) can be at the arabino(upper) or ribo(lower) position. One 2'-arabino modification is 2'-F. Other similar modifications may be made at other positions on the oligomeric compound, particularly at the 3' position of the sugar on the 3'-terminal nucleoside or at the 5' position of the 2'-5' linked oligonucleotide and 5'-terminal nucleotide. The oligomeric compound may also have a sugar mimetic, such as a cyclobutyl moiety, instead of pentofuranosyl sugar. Examples of U.S. patents disclosing the preparation of modified sugar structures include U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; and 5,576,427; This includes, but is not limited to, U.S. Patent Nos. 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920 (which are incorporated herein in their entirety by reference).

[0308] Typical sugar substituents include those described in U.S. Patent Application Publication No. 2005 / 0261218, which is incorporated herein by reference. In certain embodiments, the sugar modification is a 2'-O-Me modification, 2'F modification, 2'H modification, 2'amino modification, 4'thioribose modification, or phosphorothioate modification on the carboxyl group bonded to the 6'-carbon, or a combination thereof.

[0309] In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety containing a non-crosslinked 2'-substituent selected from F, OCH3 and OCH2CH2OCH3.

[0310] Certain modified sugar moieties include substituents that bridge two atoms of the furanosyl ring to form a second ring, resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety includes a bridge between the 4'-furanose ring atom and the 2'-furanose ring atom. Examples of such 4'-2' bridged 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 "restricted ethyl" or "cEt"), 4'-CH2-O-CH2-2', 4'-CH2-N(R)-2', 4'-CH(CH2OCH3)-O-2' ("restricted MOE" or "cMOE") and their analogues (e.g., Seth et al., U.S. Patent No. 7,399,845; Bhat et al., U.S. Patent No. 7,569,686; Swayze et al., U.S. Patent No. 7,741,457; and Swayze et al., U.S. Patent No. 8) See U.S. Patent Nos. 8,278,193), 4'-C(CH3)(CH3)-O-2' and its analogues (e.g., Seth et al., U.S. Patent No. 8,278,283), 4'-CH2-N(OCH3)-2' and its analogues (e.g., Prakash et al., U.S. Patent No. 8,278,425), 4'-CH2-ON(CH3)-2' (e.g., Allerson et al., U.S. Patent Nos. 7,696,345 and Allerson et al., U.S. Patent No. 8,124,745), 4'-CH2-C(H)(CH3)-2' (e.g., Zhou et al., J. Org. Chem., 2009, 74, 118-134), 4'-CH2-C(=CH2)-2' and its analogues (e.g., Seth et al., U.S. Patent No. 8,278,426), 4'-C(R a R b )-N(R)-O-2', 4 , -C(R a R b )-ON(R)-2', 4'-CH2-ON(R)-2', and 4'-CH2-N(R)-O-2' (wherein each R, R a and R b These are independently H, a protecting group, or C1-C 12which is alkyl (see, e.g., Imanishi et al., U.S. Patent No. 7,427,672).

[0311] In certain embodiments, such 4'-2' linkages independently comprise from 1 to 4 linked groups selected from -[C(R a )(R b )] n -, -[C(R a )(R b )] n -O-, -C(R a )=C(R b )-, -C(R a )=N-, -C(=NR a )-, -C(=O)-, -C(=S)-, -O-, -Si(R a )2-, -S(=O) x -, and -N(R a )-; wherein x is 0, 1, or 2; n is 1, 2, 3, or 4; each R a and R b is independently H, a protecting group, hydroxyl, C1-C 12 alkyl, substituted C1-C 12 alkyl, C2-C 12 alkenyl, substituted C2-C 12 alkenyl, C2-C 12 alkynyl, substituted C2-C 12 alkynyl, C5-C 20 aryl, substituted C5-C 20 aryl, a heterocyclic radical, a substituted heterocyclic radical, heteroaryl, a substituted heteroaryl, a C5-C7 cycloaliphatic radical, a substituted C5-C7 cycloaliphatic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), a substituted acyl, CN, sulfonyl (S(=O)2-J1) or sulfoxyl (S(=O)-J1); each J1 and J2 is independently H, C1-C 12 alkyl, substituted C1-C 12 alkyl, C2-C 12 alkenyl, substituted C2-C 12 alkenyl, C2-C12 Alkinyl substitution C2-C 12 Alkinyl, C5-C 20 Aryl, substitution C5-C 20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-C 12 Aminoalkyl, substituted C1-C 12 It is an aminoalkyl group or a protecting group.

[0312] The additional bicyclic sugar moiety is 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, 2017, 129, 8362-8379; Wengel et al., U.S. Patent No. 7,053,207; Imanishi et al., U.S. Patent No. 6,268,490; Imanishi et al., U.S. Patent No. 6,770,748; Imanishi et al., U.S. Patent No. RE44,779; Wengel et al., U.S. Patent No. 6,794,499; Wengel et al., U.S. Patent No. 6,670,461; Wengel et al., U.S. Patent No. 7,034,133; Wengel et al., U.S. Patent No. 8,080,644; Wengel et al., U.S. Patent No. 8,034,909; Wengel et al., U.S. Patent No. 8,153,365; Wengel et al., U.S. Patent No. 7,572,582; and Ramasamy et al., U.S. Patent No. 6,525,191; Torsten et al., International Publication No. 2004 / 1 06356; Wengel et al., International Publication No. 1999 / 014226; Seth et al., International Publication No. 2007 / 134181; Seth et al., U.S. Patent No. 7,547,684; Seth et al., U.S. Patent No. 7,666,854; Seth et al., U.S. Patent No. 8,088,746; Seth et al., U.S. Patent No. 7,750,131; Seth et al., U.S. Patent No. 8,030,467; Set h et al., U.S. Patent No. 8,268,980; Seth et al., U.S. Patent No. 8,546,556; Seth et al., U.S. Patent No. 8,530,640; Migawa et al., U.S. Patent No. 9,012,421; Seth et al., U.S. Patent No. 8,501,805; and U.S. Patent Publication Nos. (Allerson et al.) US2008 / 0039618 and (Migawa et al.) US2015 / 0191727.

[0313] In certain embodiments, the bicyclic sugar moiety and the nucleoside incorporating such a bicyclic sugar moiety are further defined by isomer configuration. For example, an LNA nucleoside (as described herein) may have an α-L configuration or a β-D configuration. [ka]

[0314] α-L-methyleneoxy(4'-CH2-0-2') or α-L-LNA bicyclic nucleosides have been incorporated into oligonucleotides exhibiting antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). In this specification, the general description of bicyclic nucleosides includes both isomer configurations. Where the position of a particular bicyclic nucleoside (e.g., LNA or cEt) is specified in the exemplary embodiments herein, they are β-D configurations unless otherwise specified.

[0315] In certain embodiments, the modified sugar moiety comprises one or more non-crosslinked sugar substituents and one or more crosslinked sugar substituents (e.g., 5'-substituted sugars and 4'-2'-crosslinked sugars).

[0316] In certain embodiments, the modified sugar moiety is a sugar substitute. In certain such embodiments, the oxygen atom of the sugar moiety is replaced with, for example, a sulfur, carbon, or nitrogen atom. In certain such embodiments, such modified sugar moiety also includes the crosslinked and / or non-crosslinked substituents described herein. For example, certain sugar substitutes include substitutions of a 4'-sulfur atom and the 2' position (see, for example, Bhat et al., U.S. Patent No. 7,875,733 and Bhat et al., U.S. Patent No. 7,939,677) and / or the 5' position.

[0317] In certain embodiments, the sugar substitute comprises a ring having more than five atoms. For example, in certain embodiments, the sugar substitute comprises a six-membered tetrahydropyran ("THP"). Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acids ("HNA"), anitol nucleic acids ("ANA"), mannitol nucleic acids ("MNA") (see, e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoroHNA: [ka]

[0318] Nucleosides comprising ("F-HNA," e.g., Swayze et al., U.S. Patent No. 8,088,904; Swayze et al., U.S. Patent No. 8,440,803; Swayze et al., U.S. Patent No. 8,796,437; and Swayze et al., U.S. Patent No. 9,005,906; F-HNA may also be called F-THP or 3'-fluorotetrahydropyran), and additional modified THP compounds having the following formulas: [ka] Here, independently of each of the modified THP nucleosides: Bx is the nucleic acid base portion; T3 and T4 are independently internucleoside linking groups that link a modified THP nucleoside to the remainder of an oligonucleotide, or one of T3 and T4 is an internucleoside linking group that links a modified THP nucleoside to the remainder of an oligonucleotide, and the other of T3 and T4 is H, a hydroxyl protecting group, a linking conjugate group, or a 5' or 3' terminal group; q1, q2, q3, q4, q5, q6, and q7 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; R1 and R2 are each independently selected from hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN (wherein X is O, S, or NJ1, and each J1, J2, and J3 is independently H or C1-C6 alkyl).

[0319] In certain embodiments, modified THP nucleosides are provided in which q1, q2, q3, q4, q5, q6, and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is not H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is methyl. In certain embodiments, modified THP nucleosides are provided in which one of R1 and R2 is F. In certain embodiments, R1 is F and R2 is H, in certain embodiments, R1 is methoxy and R2 is H, and in certain embodiments, R1 is methoxyethoxy and R2 is H.

[0320] In certain embodiments, the sugar substitute comprises a ring having more than five atoms and more than one heteroatom. For example, their use in nucleosides and oligonucleotides containing a morpholino sugar moiety has been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. Patent No. 5,698,685; Summerton et al., U.S. Patent No. 5,166,315; Summerton et al., U.S. Patent No. 5,185,444; and Summerton et al., U.S. Patent No. 5,034,506). As used herein, the term “morpholino” means a sugar substitute having the following structure: [ka]

[0321] In certain embodiments, morpholino can be modified, for example, by adding or changing various substituents from the morpholino structure described above. Such sugar substitutes are referred to herein as “modified morpholino.” In some embodiments, morpholino or modified morpholino may further include modified skeletons such as thiomorpholino or phosphorodiamidate morpholino (PMO), which are morpholino nucleosides linked by thiophosphoramidate or phosphorodiamidate nucleotide bonds.

[0322] In certain embodiments, the sugar substitute includes an acyclic moiety. Examples of nucleosides and oligonucleotides containing such acyclic sugar substitutes include, but are not limited to, peptide nucleic acids ("PNA"), acyclic butyl nucleic acids (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and nucleosides and oligonucleotides described by Manoharan et al., International Publication No. 2011 / 133876.

[0323] Many other bicyclic and tricyclic sugars and sugar substitute ring systems that can be used in modified nucleosides are known in the art.

[0324] Nucleoside residues of oligonucleotides in functional or cyclization domains can be linked to one another by any of a number of known internucleoside bonds. Two main classes of internucleoside linkages are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside bonds include, but are not limited to, phosphodiester bonds ("P=O") containing phosphophosphates (also called unmodified or naturally occurring bonds), phosphotryesters, methylphosphonates, phosphoramidates, and phosphorothioates ("P=S") and phosphorodithioates ("HS-P=S"). Typical non-phosphorus-containing internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters, thionocarbamates (-OC(=O)(NH)-S-), siloxanes (-O-SiH2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Methods for preparing phosphorus-containing and phosphorus-free internucleoside links are well known to those skilled in the art.

[0325] Such nucleoside bonds include, but are not limited to, phosphodiesters, phosphorothioates, phosphorodithioates, methylphosphonates, alkylphosphonates, alkylphosphonothioates, phosphotryesters, phosphoramidates, siloxanes, carbonates, carbolokics, acetamidates, carbamates, morpholino, borano, thioethers, cross-linked phosphoramidates, cross-linked methylenephosphonates, cross-linked phosphorothioates, and sulfone nucleoside bonds. In some embodiments, the synthetic antisense oligonucleotides of the present invention may comprise combinations of nucleotide bonds. In some embodiments, the synthetic antisense oligonucleotides of the present invention may comprise combinations of nucleotide bonds of phosphorothioates and phosphodiesters. In some embodiments, more than half but less than all of the nucleotide bonds are phosphorothioate nucleotide bonds. In some embodiments, all of the nucleotide bonds are phosphorothioate nucleotide bonds.

[0326] Modified oligonucleotides containing nucleoside-nucleoside bonds with chiral centers can be prepared as a population of modified oligonucleotides containing sterically random nucleoside-nucleoside bonds, or as a population of modified oligonucleotides containing phosphorothioate bonds, particularly stereochemical configurations. In certain embodiments, the population of modified oligonucleotides contains phosphorothioate nucleoside-nucleoside bonds, all of which are sterically random. Such modified oligonucleotides can be produced using synthetic methods that result in a random selection of the stereochemical configuration of each phosphorothioate bond. Nevertheless, as will be well understood by those skilled in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereochemical configuration. In certain embodiments, the population of modified oligonucleotides is enriched with modified oligonucleotides containing one or more specific phosphorothioate nucleoside-nucleoside bonds of specific independently selected stereochemical configurations.

[0327] In certain embodiments, the phosphorothioate bond may be a mixture of Rp and Sp enantiomers, or they may be stereoregular or substantially stereoregular in either the Rp or Sp form. In embodiments where the bond is a mixture of Rp and Sp enantiomers, the Rp and Sp forms may be located at predetermined locations within the oligonucleotide, or they may be randomly distributed throughout the oligonucleotide.

[0328] As used herein, “nucleic acid sequence” means the sequence of consecutive nucleic acid bases in a nucleic acid or oligonucleotide, independently of any alteration of sugar or nucleoside bonds.

[0329] As used herein, “nucleoside” means a compound comprising a nucleic acid base and a sugar moiety. The nucleic acid base and sugar moiety are either unmodified or modified, independently of each other. As used herein, “modified nucleoside” means a nucleoside comprising a modified nucleic acid base and / or a modified sugar moiety. Modified nucleosides include debasic nucleosides that lack a nucleic acid base. “Linked nucleosides” are nucleosides linked in a continuous sequence (i.e., there are no additional nucleosides between linked nucleosides).

[0330] The term "nucleic acid" in its broadest sense includes any compound and / or substance containing polymers of nucleotides. These polymers are often called oligonucleotides or polynucleotides. Exemplary nucleic acids of the present invention include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA (including LNA having a β-D-ribo structure, α-LNA having an α-ribo structure (α-diastereomer of LNA), 2'-amino-LNA having 2'-amino functionalization, and 2'-amino-α-LNA having 2'-amino functionalization)) or hybrids thereof.

[0331] As used herein, “oligomer compound” means an oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group. The oligomer compound may or may not be paired with a second oligomer compound complementary to the first oligomer compound. “Single-stranded oligomer compound” is an unpaired oligomer compound.

[0332] As used herein, “oligonucleotide” means a chain of linked nucleosides of any length containing polynucleotides, linked via nucleoside bonds, where each nucleoside and nucleoside bond may be modified or unmodified. Unless otherwise indicated, oligonucleotides consist of 8 to 50 linked nucleosides.

[0333] As used herein, “modified oligonucleotide” means an oligonucleotide in which at least one nucleoside or nucleoside bond has been modified. As used herein, “unmodified oligonucleotide” means an oligonucleotide that does not contain any nucleoside modification or nucleoside modification.

[0334] As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use when administered to an animal. Certain such carriers enable the formulation of pharmaceutical compositions, for example, as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and lozenges for oral administration by a subject. In certain embodiments, the pharmaceutically acceptable carrier or diluent is sterile water; sterile saline; or sterile buffer solution.

[0335] As used herein, “pharmaceutically acceptable salt” means a physiologically and pharmaceutically acceptable salt of a compound, such as an oligomeric compound, i.e., a salt that retains the desired biological activity of the parent compound and does not impart any undesirable toxicological effects.

[0336] As used herein, “pharmaceutical composition” means a mixture of substances suitable for administration to a subject. For example, a pharmaceutical composition may include an antisense compound and a sterile aqueous solution. In certain embodiments, the pharmaceutical composition exhibits activity in a free uptake assay in a specific cell line.

[0337] As used herein, “phosphorus moiety” means a group of atoms containing a phosphorus atom. In certain embodiments, the phosphorus moiety includes mono-, di-, or tri-phosphorus phosphates or phosphorothioates.

[0338] As used herein, “polypeptide” means a polymer of amino acid residues (natural or unnatural) that are most often linked by peptide bonds. As used herein, the term refers to proteins, polypeptides, and peptides of any size, structure, or function. In some examples, the encoded polypeptide is smaller than about 50 amino acids, and therefore the polypeptide is called a peptide. If a polypeptide is a peptide, it is 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, orthologues, paralogs, fragments, and other equivalents, variants, and analogs of the above. Polypeptides may be single molecules or multimolecular complexes such as dimers, trimers, or tetramers. They may also contain single-chain or polychain polypeptides such as antibodies or insulin, and may associate or link. Most commonly, disulfide bonds are found in polychain polypeptides. The term polypeptide may also apply to amino acid polymers, in which one or more amino acid residues are artificial chemical analogs of corresponding natural amino acids.

[0339] The term "polypeptide variant" refers to a molecule whose amino acid sequence differs from that of its native or reference sequence. Amino acid variants may have substitutions, deletions, and / or insertions at specific positions within the amino acid sequence compared to the native or reference sequence. Typically, variants have at least about 50% identity (homology) to the native or reference sequence, preferably at least about 80%, and more preferably at least about 90% identity (homology) to the native or reference sequence.

[0340] As used herein, “prodrug” means an in vitro therapeutic agent that is converted into a different form within an animal or its cells. Typically, the conversion of a prodrug within an animal is facilitated by the action of enzymes (e.g., endogenous or viral enzymes) or chemicals present in the cells or tissues and / or by physiological conditions.

[0341] As used herein, "OMe" means methoxy. "2'-OMe" means a 2'-OCH3 group instead of a 2'OH group in the ribosyl sugar moiety.

[0342] The enumeration of numerical ranges by endpoints includes all numbers and fractions contained within each range, as well as the enumerated endpoints.

[0343] The ranges provided herein are understood to be abbreviated representations of all values ​​within that range. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subrange 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 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested subranges" extending from any endpoint of the range are particularly intended. For example, nested subranges of the exemplary range 1-50 may include 1-10, 1-20, 1-30, and 1-40 in one direction, or 50-40, 50-30, 50-20, and 50-10 in the other direction.

[0344] As used herein, “reduce or inhibit quantity or activity” means a reduction or blockage of transcriptional expression or activity compared to the untreated or control sample, and does not necessarily mean complete elimination of transcriptional expression or activity.

[0345] As used herein, “self-complementary” with respect to oligonucleotides means oligonucleotides that hybridize at least partially with themselves.

[0346] As used herein, “standard cell assay” means the assay described in Example 1 and reasonable variations thereof.

[0347] As used herein, “sterically random chiral centers” in the context of a group of molecules of the same molecular formula means chiral centers having a random stereochemical configuration. For example, in a group of molecules containing sterically random chiral centers, the number of molecules having a sterically random (S) configuration of a chiral center may be the same as, but not necessarily the same as, the number of molecules having a sterically random (R) configuration of a chiral center. The stereochemical configuration of a chiral center can be considered random if it is the result of a synthetic method not designed to control the stereochemical configuration. In certain embodiments, a sterically random chiral center is a sterically random phosphorothioate nucleoside bond.

[0348] As used herein, “sugar moiety” means an unmodified sugar moiety or a modified sugar moiety. As used herein, “unmodified sugar moiety” means a 2'-OH(H) furanosyl moiety, such as that found in RNA (“unmodified RNA sugar moiety”), or a 2'-H(H) moiety, such as that found in DNA (“unmodified DNA sugar moiety”). An unmodified sugar moiety has one hydrogen atom at each of the 3' and 4' positions, an oxygen atom at the 3' position, and two hydrogen atoms at the 5' position. As used herein, “modified sugar moiety” or “modified sugar” means a modified furanosyl sugar moiety or sugar substitute. As used herein, a modified furanosyl sugar moiety means a furanosyl sugar that contains a non-hydrogen substituent in place of at least one hydrogen atom of an unmodified sugar moiety. In certain embodiments, the modified furanosyl sugar moiety is a 2'-substituted sugar moiety. Such modified furanosyl sugar moieties include bicyclic sugars and acyclic sugars.

[0349] As used herein, “sugar substitute” means a modified sugar moiety having a furanosyl moiety other than that which can link a nucleic acid base to another group, for example, an internucleoside bond, conjugate group, or terminal group in an oligonucleotide. Modified nucleosides containing sugar substitutes can be incorporated at one or more positions in an oligonucleotide, and such oligonucleotides can hybridize to complementary oligomeric compounds or nucleic acids.

[0350] As used herein, “target nucleic acid” and “target RNA” mean nucleic acids that are designed to be affected by an antisense compound.

[0351] As used herein, “target region” means the portion of a target nucleic acid that is designed for the oligomeric compound to hybridize.

[0352] As used herein, “terminal group” means a group of chemical groups or atoms covalently bonded to the end of an oligonucleotide.

[0353] As used herein, “therapeutic dose” means the amount of a drug that provides a therapeutic benefit to an animal. For example, a therapeutic dose improves the symptoms of a disease.

[0354] As used herein, “to treat,” “to treat,” or “to process” means to administer any of the compounds described herein to result in a modification or improvement of a disease, disorder, or symptom.

[0355] A “part” refers to a predetermined number of consecutive (i.e., linked) nucleic acid bases of a nucleic acid. In a particular embodiment, the part is a predetermined number of consecutive nucleic acid bases of the target nucleic acid. In a particular embodiment, the part is a predetermined number of consecutive nucleic acid bases of the antisense compound.

[0356] The terms "co-administration" or "to be co-administered" generally refer to the administration of at least two different substances. Co-administration refers to the simultaneous administration of at least two different substances in any order, either in single doses or separate doses, and in any order with a time interval of up to several days.

[0357] The term “combined” generally means administering an oligonucleotide compound according to the present invention with another agent useful for treating a disease or condition in which the compound does not lose its activity during the course of treating the patient. Such administrations may be carried out in any order, including simultaneous administration and sequences with time intervals ranging from a few seconds to several days. Such combined treatment may also involve more than one dose of the compound according to the present invention and / or other agents independently. The administration of the compound according to the present invention and other agents may be via the same or different routes.

[0358] The terms “individual,” “subject,” or “patient” generally refer to mammals, including humans. The term “mammal” is expressly intended to include warm-blooded vertebrates, including but not limited to humans, non-human primates, rats, mice, cattle, dogs, horses, cattle, cows, pigs, sheep, and rabbits. As used herein, “individual requiring it” refers to a human or non-human animal selected for a procedure or treatment requiring such a procedure or treatment. As used herein, “inhibit expression or activity” refers to a reduction or blockage of RNA or protein expression or activity, and does not necessarily indicate complete elimination of expression or activity. [Examples]

[0359] Synthesis of CPNs containing antisense oligonucleotide functional domains The cyclic prodrug nucleic acids according to the present invention can be synthesized by procedures well known in the art, such as phosphoramidate or H-phosphonate chemistry, which can be performed manually or by an automated synthesizer. For example, the oligonucleotides of the present invention can be synthesized by a linear synthesis approach.

[0360] The compounds used herein have been synthesized using phosphoramidite chemistry. These protocols are described in detail, for example, at pubs.rsc.org / en / content / chapter / bk9781788012096-00453 / 978-1-78801-209-6, which are incorporated herein by reference.

[0361] Inhibition of target RNA by CPNs containing antisense oligonucleotide functional domains. Cyclic prodrug nucleic acids targeting PCSK9 nucleic acid can be designed, and their effects on PCSK9 mRNA can be tested in vitro.

[0362] Hepa 1-6 cells can be cultured in DMEM medium + 10% FBS and 100 U / ml Pen / Strep (cells from ATCC). Cells can be seeded and incubated overnight until approximately 70% confluent at transfection—approximately 100,000 cells / ml / 12-well plate. The cell medium can be changed, with 900 μl added to each well. Oligonucleotides can be mixed with lipofectamine in Opti-MEM medium and added to the lipids (1:1 ratio), and incubated for 15-20 minutes. 100 μl at a 100 nM antisense concentration can be added to each well. After a processing period of approximately 16-48 hours, cells can be harvested for RNA and / or protein analysis.

[0363] The culture supernatant can be assayed for AK-releasing cytotoxicity. Taqman probes can be used for mPCSK9 and PPIB or HPRT1 (housekeeping control) (probes provided by ThermoFisher).

[0364] Cyclic prodrug nucleic acids targeting PNPLA3 nucleic acid can be designed, and their effects on PNPLA3 mRNA can be tested in vitro.

[0365] Human HepG2 cells can be cultured according to ATCC recommended conditions and medium (Eagle's Minimum Essential Medium containing 10% FBS). Cells can be plated at a density of 50K / well into a PDL-coated 96-well plate and reverse transfected with 0.6 ul / well of RNAiMax and oligonucleotide compounds at the indicated concentrations. After 24 hours, cells can be harvested using Cells to CT lysis reagent (ThermoFisher 4391851C). RNA can be reverse transcribed into a cDNA template using the RT reagent kit (ThermoFisher A39110). Quantitative PCR can be performed using qPCR Master Mix (ThermoFisher 4444964). PNPLA3 expression levels can be quantified using the PNPLA3-FAM probe (ThermoFisher 4351368 assay ID: Hs00228747) and normalized by the housekeeping gene POLR2A (ThermoFisher 4448491 assay ID: Hs01108291). The data can be analyzed using GraphPad Prism.

[0366] To investigate whether cyclization of linear ASOs using split-merized modified RNA can offer further efficacy, the levels of PNPLA3 knockdown can be compared using oligonucleotides with the same nucleotide sequence in different split-merized and chemical sequences.

[0367] Cyclic prodrug nucleic acids targeting APOC3 nucleic acid were designed, and their effects on APOC3 mRNA were tested in vitro. Hep3B cells (of human liver origin) were cultured according to ATCC recommended conditions and medium (Eagle's Minimum Essential Medium containing 10% FBS). Cells were seeded at a density of 20 K / well in 96-well plates and reverse-transfected with 0.3 ul / well of RNAiMax and nine different concentrations (20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, 0.000256, and 0.00001 nM) of antisense oligonucleotides (ASOs). Control cells were transfected with 0.3 ul / well of RNAiMax and medium alone or with 25 nM non-template control ASO, and incubated for 48 hours post-transfection. Gene expression assays were performed using the Invitrogen® TaqMan® Fast Advanced Cells-to-CT® Kit, following the manufacturer's protocol (A35378, Invitrogen®). Briefly, cells were harvested using Cells to CT lysis reagent, and total RNA was immediately reverse transcribed to cDNA using RT reagent. Quantitative polymerase chain reaction (qPCR) was performed in multiplexed reactions using the qPCR Fast Advanced Master Mix and pre-designed primers and FAM-labeled probe (APOC3; Hs00163644_m1), and normalized using pre-designed primers and VIC-labeled probe (HPRT1; Hs02800695_m1) for the reference gene hypoxanthine-guanine phosphoribosyltransferase (HPRT1). Data were plotted and analyzed using the GraphPad Prism "Absolute IC50" nonlinear regression dose-response model. The results are shown in Figures 2A to 2J.

[0368] We designed cyclic prodrug nucleic acids targeting MAPT nucleic acids and tested their effects on MAPT mRNA in vitro. U-251 MG cells were cultured according to ATCC recommended conditions and medium (Eagle's Minimum Essential Medium with 10% FBS). Cells were seeded at a density of 20 K / well in 96-well plates and reverse-transfected with 0.3 µl / well of RNAiMax and six different concentrations (25, 6.25, 1.56, 0.39, 0.10, and 0.024 nM) of cyclic structured antisense oligonucleotides (CSOs). Control wells were transfected with 0.3 µl / well of RNAiMax and medium only or with 25 nM non-template control antisense oligonucleotide and incubated for 48 hours post-transfection. Gene expression was assayed using the Invitrogen® TaqMan® Fast Advanced Cells-to-CT® Kit according to the manufacturer's protocol (A35378, Invitrogen®). Briefly, cells were harvested using Cells to CT lysis reagent, and immediately afterward, total RNA was reverse transcribed into cDNA using RT reagent. Quantitative polymerase chain reaction (qPCR) was performed in multiplexed reactions using qPCR Fast Advanced Master Mix and pre-designed primers and FAM-labeled probes (4351370, Invitrogen®) for the corresponding target gene (GOI). The results were normalized using pre-designed primers and VIC-labeled probes (#4448486, Invitrogen®) for the reference gene hypoxanthine-guanine phosphoribosyltransferase (HPRT1). The data were plotted and analyzed using the "Absolute IC50" nonlinear regression dose-response model in GraphPad Prism. The results are shown in Figures 3A to 3D.

[0369] Generation of immune response by CPNs containing immunostimulatory oligonucleotide functional domains Mouse splenocyte restimulation assay Spleens from C57BL / 6J mice can be mechanically dispersed into a single-cell suspension using ammonium chloride lysis buffer (catalog number 420302, BioLegend, San Diego, CA), which is used to remove red blood cells. Cell viability can be determined using vital dye staining and an automated cell counting system (Count 3, Thermo Fisher, Waltham, MA). After counting, 1 x 10⁻⁶ cells are used. 5 Individual viable mouse splenocytes can be seeded into each well of a 96-well flat-bottom sterile tissue culture plate (catalog number 3596, Corning, Glendale, AZ) containing RPMI 1640 (catalog number A1049101, Thermo Fisher) with 10% fetal bovine serum (catalog number F2442, MilliporeSigma, Burlington, MA).

[0370] Immunostimulatory oligonucleotide (ISO) agonists for Toll-like receptor 9 (TLR9) and CPNs containing the TLR9 immunostimulatory oligonucleotide (ISO) functional domain can be synthesized using standard methods 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 min). After annealing, the sample concentration can be determined using absorbance for oligonucleotide concentration determination using a NanoDrop spectrophotometer (Thermo Fisher). A control TLR9 agonist may be used. A dose of CPN or control agonist can be added to the indicated wells, and cells can be stimulated in a 37°C, 5% CO2 incubator for 24 hours. After the incubation period, the cell culture supernatant can be collected and analyzed using a custom multiple cytokine / chemokine assay kit (U-PLEX custom biomarker assay, catalog number K15069M-2, Meso Scale Diagnostics, Rockville, MD). Data collection is performed using an MSD S600 bioanalyzer (Meso Scale), and raw data analysis is performed using MSD Discovery Workbench (Meso Scale).

[0371] To evaluate the activity of CPNs containing an immune antagonist oligonucleotide functional domain, such CPNs can be incubated alone or with a TLR9 agonist. Cytokines can be evaluated after 24 hours.

[0372] Although the present invention has been specifically shown and described with reference to its preferred embodiments, it will be understood by those skilled in the art that various modifications in form and detail can be made without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. A cyclic prodrug nucleic acid (CPN) comprising a functional domain and a cyclic domain, wherein the functional domain comprises an oligonucleotide, the cyclic domain comprises a first nucleic acid molecule and a second nucleic acid molecule, the 5' end of the oligonucleotide of the functional domain is ligated to the first nucleic acid molecule, and the 3' end of the oligonucleotide of the functional domain is ligated to the second nucleic acid molecule; the first nucleic acid molecule and the second nucleic acid molecule are independently 6 to 50 nucleotides long, the nucleotides of the first nucleic acid molecule and the second nucleic acid molecule are independently selected from RNA or DNA, and the first nucleic acid molecule and the second nucleic acid molecule are complementary to each other, have opposite polarities, and hybridize to form a double-stranded portion.

2. The CPN has a structure according to formula I or formula II: 3'-Y ｎ Y ６ Y ５ Y ４ Y ２ Y ２ Y １ -5'-5'-functional domain oligonucleotide-3'-5'-X １ X ２ X ３ X ４ X ５ X ６ X ｍ -3' Equation I 5'-Y １ Y ２ Y ３ Y ４ Y ５ Y ６ Y ｎ -3'-5'-Functional domain oligonucleotide-3'-3'-X ｍ X ６ X ５ X ４ X ３ X ２ X １ -5' Formula II It has, During the ceremony, Y １ ~Y ｎ is the first nucleic acid molecule as defined herein; X １ ~X ｍ is the second nucleic acid as defined herein; n is 0 to 44; and m is between 0 and 44. The CPN according to claim 1.

3. A CPN according to claim 1 or 2, comprising an antisense oligonucleotide 17 to 25 nucleotides in length, and comprising a functional domain containing at least 12 consecutive nucleic acid bases complementary to an equal-length portion of the target RNA sequence, wherein the antisense oligonucleotide compound comprises a 3' domain and a 5' domain adjacent to the 3' domain. The 3' domain begins with the 3' terminal nucleotide, is 10 to 12 nucleotides long, and each nucleotide is independently any deoxyribonucleotide, and each nucleotide bond between adjacent deoxyribonucleotides is a phosphodiester or phosphorothioate nucleotide bond or a combination thereof; The 5' domain begins with the first nucleotide following the 3' domain and continues to the terminal nucleotide of the 5' end, wherein the 5' domain comprises an unmodified deoxyribonucleotide, an unmodified ribonucleotide, a modified deoxyribonucleotide, a modified ribonucleotide, or a combination thereof, wherein at least three nucleotides of the 5' domain comprise a modified deoxyribonucleotide or modified ribonucleotide containing a modified sugar and / or backbone, wherein the 5' domain comprises a modified deoxyribonucleotide or modified ribonucleotide.

4. The CPN according to claim 3, wherein the 3' domain is 12 nucleotides long and includes nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 from the 3' end.

5. The CPN according to claim 3, wherein the 3' domain is 11 nucleotides long and includes nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 from the 3' end.

6. The CPN according to claim 3, wherein the 3' domain is 10 nucleotides long and includes nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 from the 3' end.

7. The CPN according to any one of claims 1 to 6, wherein the first nucleic acid sequence is DNA and the second nucleic acid sequence is RNA.

8. The CPN according to any one of claims 1 to 6, wherein the first nucleic acid sequence is RNA and the second nucleic acid sequence is DNA.

9. The CPN according to claim 7, wherein the nucleotide of the first nucleic acid sequence comprises an unmodified deoxyribonucleotide, and the nucleotide of the second nucleic acid sequence comprises an unmodified ribonucleotide.

10. The CPN according to claim 8, wherein the nucleotide of the first nucleic acid sequence comprises an unmodified ribonucleotide, and the nucleotide of the second nucleic acid sequence comprises an unmodified deoxyribonucleotide.

11. The CPN according to any one of claims 3 to 10, wherein at least half of the nucleotides in the 5' domain are modified deoxyribonucleotides or modified ribonucleotides comprising a modified sugar and / or skeleton.

12. The CPN according to any one of claims 3 to 11, wherein all of the nucleotides in the 5' domain are modified deoxyribonucleotides or modified ribonucleotides comprising a modified sugar and / or skeleton.

13. The CPN according to any one of claims 1 to 12, wherein the 5' end of the first nucleic acid molecule is linked to the 5' end of the oligonucleotide, and the 5' end of the second nucleic acid molecule is linked to the 3' end of the oligonucleotide.

14. The CPN according to any one of claims 1 to 12, wherein the 3' end of the first nucleic acid molecule is linked to the 5' end of the oligonucleotide, and the 3' end of the second nucleic acid molecule is linked to the 3' end of the oligonucleotide.

15. The CPN according to any one of claims 3 to 14, wherein the 5' domain comprises a modified ribonucleotide, and the modified ribonucleotide comprises a 2' substituted ribonucleotide.

16. The CPN according to claim 15, wherein the 2'-substituted ribonucleotide is a 2'-OMe ribonucleotide or a 2'-MOE ribonucleotide.

17. The CPN according to any one of claims 1 to 16, wherein the internucleotide bonds of the oligonucleotide of the functional domain and / or the first nucleic acid and the second nucleic acid of the CPN include phosphorothioate internucleotide bonds, phosphodiester internucleotide bonds, or a combination thereof.

18. The CPN according to any one of claims 1 to 17, wherein the internucleotide bonds between the first nucleic acid and the second nucleic acid of the CPN include phosphorothioate internucleotide bonds.

19. The CPN according to any one of claims 1 to 17, wherein the internucleotide bonds between the first nucleic acid and the second nucleic acid of the CPN include phosphodiester internucleotide bonds.

20. The CPN according to any one of claims 1 to 19, wherein the oligonucleotide is linked to the first nucleic acid molecule and the second nucleic acid molecule via direct bonding.

21. The CPN according to any one of claims 1 to 20, wherein the first nucleic acid molecule and the second nucleic acid molecule of the cyclic domain hybridize with each other to form a cyclic structure.

22. The CPN according to any one of claims 1 to 21, wherein both the first nucleic acid molecule and the second nucleic acid molecule are not DNA.

23. The CPN according to any one of claims 1 to 22, wherein the double-stranded portion formed by the first nucleic acid molecule and the second nucleic acid molecule is cleaved in situ to linearize the cyclic structure, thereby presenting the oligonucleotide of the functional domain and enabling it to perform its function.

24. A pharmaceutical composition comprising a CPN and a pharmaceutically acceptable carrier as described in any one of claims 1 to 23.

25. The pharmaceutical composition according to claim 24, further comprising one or more agents selected from small molecules, peptides, vaccines, antigens, antibodies, cytotoxic agents, kinase inhibitors, allergens, antibiotics, siRNA molecules, antisense oligonucleotides, TLR antagonists, chemotherapeutic agents, targeted therapeutic agents, activated cells, proteins, gene therapy vectors, peptide vaccines, protein vaccines, DNA vaccines, adjuvants, and costimulatory molecules, or combinations thereof.

26. A method for inhibiting gene expression, comprising administering CPN according to any one of claims 1 to 23 or the composition according to claim 24 or 25.

27. A method for treating a target disease or disorder, wherein modulating RNA is beneficial for treating the target, and the method comprises administering a CPN according to any one of claims 1 to 23 or a composition according to claim 24 or 25.

28. A method for treating a target disease or disorder, wherein inhibiting gene expression is beneficial for treating the target, and the method comprises administering a CPN according to any one of claims 1 to 23 or a composition according to claim 24 or 25.

29. A method for treating a target disease or disorder, wherein inducing an immune response is beneficial for treating the target, the method comprising administering a CPN according to any one of claims 1 to 23 or a composition according to claim 24 or 25.

30. A method for treating a target disease or disorder, wherein inhibiting an immune response is beneficial for treating the target, and the method comprises administering a cyclic CPN according to any one of claims 1 to 23 or a composition according to claim 24 or 25.

31. A method for inducing a nonsense mutation-dependent mRNA degradation mechanism of a target RNA, comprising administering a CPN according to any one of claims 1 to 23 or a composition according to claim 24 or 25.

32. A method for increasing the level of mRNA encoding a protein or functional mRNA, and for increasing the expression of the protein or functional mRNA, comprising administering a CPN according to any one of claims 1 to 23 or a composition according to claim 24 or 25.

33. The method according to any one of claims 26 to 28, 31, or 32, which is useful for treating subjects having a disease or disorder in which the regulatory expression of a gene is beneficial.

34. The method according to claim 33, wherein the disease or disorder is caused by abnormal expression or product of a cellular gene.

35. The method according to any one of claims 26 to 34, wherein the CPN is administered locally.

36. The CPN according to claim 1 or 2, wherein the functional domain is selected from antisense oligonucleotides, microRNAs (miRNAs), siRNAs, piRNAs, hnRNAs, ncRNAs, snRNAs, miRNA mimetics, sgRNAs, esiRNAs, shRNAs, lncRNAs, mRNAs, adeno-associated viruses (AAVs), guide RNAs for CRISPR-based systems, ADAR mobilizing RNAs for RNA-acting adenosine deaminase (ADAR) systems, splicing oligonucleotides, aptamers, immunostimulatory oligonucleotides, or immunoinhibitory oligonucleotides.

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