Asymmetric short double-stranded RNAs interspersed with deoxyribonucleotides and their uses as gene silencing techniques

Abstract

The present invention discloses a novel type of gene silencing technology for modulating target nucleic acid and / or protein levels in cells, tissues, organisms, and animals. This new technology provides compositions for use in gene targeting or gene silencing applications, including the prevention and treatment of human diseases. The compositions include asymmetric, short, double-stranded RNA molecules in which the sense strand is shorter than the antisense strand. The double-stranded RNA molecules further include at least one deoxyribonucleotide monomer interspersed segment. The present invention also provides methods for using the compositions to modulate the expression or function of target genes, or for the treatment or prevention of diseases, as well as for other medical or biological applications.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of co-pending U.S. Provisional Patent Application No. 63 / 431,154, filed December 8, 2022, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention relates to a novel type of gene silencing technology based on asymmetric short double-stranded RNA interspersed with deoxyribonucleotides, and also to related compositions and methods that can be used in biological or medical research, in the treatment and prevention of diseases, and for the application of gene silencing in other fields of biology. [Background technology]

[0003] Background of the Invention Modern medical therapeutics rely on two fundamental technologies: small molecule chemistry and protein / antibody technology. However, only approximately 10% of targets identified through genomic and biomedical research can be addressed by these two fundamental technologies. Oligonucleotides have the potential to address a vast number of targets, including those that are not druggable by small molecule chemistry and antibody / protein technology. Over 40 years of research has led to the development of antisense oligonucleotide (ASO) and small interfering RNA (siRNA) technologies (Cy A. Stein et al., 2017). However, despite these decades of research, significant druggability issues have prevented ASO and siRNA technologies from becoming mainstream therapeutic platforms, except for a few clinical orphan indications. These druggability issues include, among others, low silencing efficiency, off-target effects, stimulation of unintended immune responses, tissue penetration challenges, and in vivo delivery. Therefore, there is a significant unmet need to create novel technologies to target genes of interest in various biological and medical applications.

[0004] ASO is a gene silencing technology based on a concept first proposed in 1978 (Zamecnik PC et al., 1978). In general, the principle behind ASO technology is that antisense oligonucleotides hybridize to target nucleic acids and modulate gene expression activity or function, for example, transcription / post-transcription or translation. The mechanisms can be broadly categorized as follows: (1) simple occupancy, in which ASO binding leads to translation arrest, splicing inhibition, or introduction of alternatively spliced ​​variants, without promoting RNA degradation; (2) occupancy-induced destabilization, in which ASO binding promotes RNA degradation by endogenous enzymes such as ribonuclease H1 (RNase H1); and (3) translational modulation: ASOs can block other inhibitory or regulatory elements in upstream open reading frames (uORFs) or 5'UTRs, thereby increasing or modulating translation efficiency (Stanley T. Crooke et al., 2008; C. Frank Bennett, 2010; Richard G. Lee, 2013; Stanley T. Crooke, 2017). ASO structures are single-stranded deoxyribonucleotide sequences that can bind to target RNAs through base pairing. After 40 years of research, ASO technology has been improved by various chemical modifications of single-stranded oligonucleotides, such as phosphorothioate substitutions or other modified nucleotides (Iwamoto N et al. 2017, Crooke ST, 2017; Crooke ST et al., 2018; see U.S. Patent Nos. 7,919,472 and 9,045,754).

[0005] RNAi, a mechanism by which short double-stranded RNAs induce the loss of homologous RNAs, was first observed in plants and demonstrated in the nematode Caenorhabditis elegans (A. Fire et al., 1998). This mechanism involves the degradation of long dsRNAs into short interfering double-stranded RNAs (siRNAs) and the interaction of siRNAs with the multiprotein RNA-induced silencing complex (RISC). Within RISC, the siRNAs unwind, discarding the sense strand, while the antisense or guide strand binds to the RISC endonuclease AGO2, which then cleaves the target RNA (de Fougerolles et al., 2007; Ryszard Kole, 2016). RNAi is a sequence-specific post-transcriptional gene silencing process triggered by short double-stranded RNAs in the cytoplasm and is therefore used to silence cytoplasmic mRNAs. In mammalian cells, synthetic siRNAs or asymmetric short interfering RNAs (aiRNAs or asymmetric siRNAs) can be used to induce gene silencing through an RNAi RISC-dependent mechanism (Elbashir SM et al., 2001; Sun X et al., 2008; see U.S. Patent Nos. 7,056,704 and 9,328,345).

[0006] Oligonucleotides have been studied for decades and are believed to have great potential to become an entirely new class of therapeutics. However, their limited silencing efficiency, delivery challenges, and dose-dependent adverse effects, including hybridization-dependent and hybridization-independent toxicities, continue to limit these novel therapeutics (C. Frank Bennett, 2010; and C. Frank Bennett, 2019; Roberts TC et al., 2020; Crooke ST et al., 2018; Setten RL et al., 2020). Generally, ASO compounds are less potent at inducing gene silencing than siRNA-based compounds, but ASO compounds offer several pharmaceutical advantages over siRNA compounds. Currently, ASOs and siRNA remain two equally important platform technologies for designing gene-silencing therapeutics (Crooke ST et al., 2018; Roberts TC et al., 2020). Hybridization-dependent toxicity of oligonucleotides is thought to be primarily due to hybridization with non-target genes ("off-target effects") (Jackson et al., 2003; Lin X et al., 2005). Hybridization-independent toxicity occurs through the interaction of oligonucleotides with proteins, and these effects include increased clotting time, proinflammatory effects, and activation of the complement pathway. These effects tend to occur at higher doses of oligonucleotides and are dose-dependent. For example, at higher concentrations, ASOs cause renal tubular changes and thrombocytopenia (Geary, RS. et al., 2007; Kwoh JT, 2008).Clinically, the main tolerability and safety issues for first-generation PS antisense oligodeoxynucleotides and second-generation 2'-MOE-modified antisense oligonucleotides have been proven to be hybridization-independent effects, such as prolonged activated partial thromboplastin time, injection site reactions, and systemic symptoms such as fever, chills, and headache (C. Frank Bennett, 2010; Henry SP, 2008; Kwoh JT, 2008). Even the most optimized ASOs have generally proven to be much less effective than siRNAs and to have uniform dose-dependent toxicity (Kendall S. Frazier, 2015). To mitigate the dose-dependent toxicity of oligonucleotides, various chemical modifications have been used over the past 40 years to overcome the limited efficacy and associated safety issues (Iwamoto N et al., 2017, Crooke ST et al., 2018; and Roberts TC et al., 2020).

[0007] Compared to ASOs, the off-target silencing effects of siRNA duplexes are thought to be mediated by sense strand-mediated silencing, competition with endogenous miRNA pathways, and interactions with TLRs or other proteins (Setten RL et al., 2019). Additionally, typical 21nt / 19bp siRNA duplexes are inefficient in terms of cell and tissue penetration and require extensive chemical modifications to enhance stability and other pharmaceutical properties. Asymmetric siRNAs (or aiRNAs) have been designed to overcome the off-target effects mediated by the sense strand of symmetric siRNAs, as well as other off-target mechanisms (Sun X et al., 2008; Grimm D, 2009; Selbly CR et al., 2010; and PCT Patent Publication WO2009029688).

[0008] In summary, after more than 40 years of innovation in ASO technology and more than 20 years of research into RNAi-based technologies, the successful development of gene-targeted therapies for nearly 90% of the targets involved in human diseases remains challenging. Furthermore, currently approved oligonucleotide drugs cost more than $500,000 per patient per year and are therefore unable to address diseases affecting the general population. Therefore, novel technologies to overcome these challenges are urgently needed. Any reference cited herein is not admitted to be prior art to the claimed invention. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 7,056,704 [Patent Document 2] U.S. Patent No. 9,328,345 Summary of the Invention [Means for solving the problem]

[0010] Summary of the Invention The present invention is based on the surprising discovery of potent gene silencing induced by asymmetric short double-stranded ribonucleotides (asdRNAs) containing deoxyribonucleotide-interspersed segments ("ISDs"). This novel type of gene silencing technology, enabled by asdRNAs interspersed with one or more deoxyribonucleotides, utilizes short double-stranded molecules composed of linked nucleotide monomers, each selected from the group of naturally occurring nucleotides, their analogs, and modified nucleotides (collectively referred to hereinafter as "nucleotide monomers"). In other words, the nucleotide monomers used in embodiments of the present invention include "ribonucleotide monomers" selected from the group of naturally occurring ribonucleotides, their analogs, and modified ribonucleotides. Furthermore, the gene silencing function of asdRNAs can be dramatically enabled or enhanced by incorporating one or a few interspersed deoxyribonucleotide monomers. The "deoxyribonucleotide monomers" can be selected from the group of naturally occurring deoxyribonucleotides, their analogs, and modified deoxyribonucleotides.

[0011] In the present invention, at least 50% of the nucleotide monomers of the asdRNA molecules of the present invention are ribonucleotide monomers, and therefore the entire molecule is referred to as a double-stranded RNA molecule, or more specifically, a short double-stranded RNA (sdRNA) molecule, or even more specifically, an asymmetric short double-stranded RNA (asdRNA) molecule. The molecules of the present invention are further interspersed with deoxyribonucleotide monomers, which form at least one deoxyribonucleotide monomer interspersed segment ("ISD").

[0012] The novel platform technology based on asdRNA contained in the present disclosure has a high gene silencing effect, and other advantages will be disclosed hereafter.In one embodiment, the sense strand of the oligonucleotide monomer and the antisense strand of the oligonucleotide monomer are substantially complementary to the target ribonucleotide sequence.The inventors' data show that the asdRNA molecules of the present invention can induce gene silencing at picomolar concentrations (for example, 800pM, 500pM, 300pM, 200pM, 100pM or even lower) due to their unique and novel composition, which is more potent than existing gene silencing technologies, and therefore allows for reduced dose-dependent toxicity. The asdRNA molecules of the present invention also have at least one of the following advantages over existing gene silencing technologies: enabling gene silencing not only in the cytoplasm but also in the nucleus(s) and mitochondria(s) (in contrast to siRNA / aiRNA-based gene silencing, which occurs only in the cytoplasm); reduced off-target effects; elimination or reduction of undesired interference with endogenous microRNA function, as observed with siRNA; better tissue penetration; better stability; lower synthesis costs; and other improved pharmaceutical properties. Therefore, the asdRNA molecules of the present invention have great potential to address various challenges faced by ASO, siRNA / aiRNA, and other existing gene silencing technologies. In addition, the asdRNA molecules of the present invention can modulate gene expression activity or function, post-transcriptional and / or translational stages, whereas RNAi can only induce gene silencing at the post-transcriptional level. Furthermore, asdRNAs can tolerate a greater and more extensive range of chemical modifications, including non-RNA-like nucleotide modifications or substitutions. The asdRNA molecules of the present invention can be used in any area where current oligonucleotides are applied or intended for use, including research, diagnostics, disease prevention and treatment, and other applications in the fields of biology, including agriculture and veterinary medicine.

[0013] In a first aspect, the present invention provides a composition comprising an asymmetric short double-stranded RNA (asdRNA) molecule having a first strand and a second strand, each of which comprises linked ribonucleotide monomers with a deoxyribonucleotide monomer interspersed segment. The ribonucleotide monomers within the molecule are selected from the group consisting of naturally occurring ribonucleotides, their analogs, and modified ribonucleotides; the deoxyribonucleotide monomer interspersed segment within the asdRNA molecule is selected from the group consisting of naturally occurring deoxyribonucleotides, their analogs, and modified deoxyribonucleotides. The asdRNA molecule is an asymmetric short double-stranded RNA (asdRNA) molecule in which the second strand is shorter than the first strand. The first strand is substantially complementary to the segment targeted by at least one targeting region of the RNA and can therefore be considered an antisense strand or antisense oligonucleotide. Furthermore, the second strand, which can be considered a sense strand or sense oligonucleotide, is substantially complementary to the first strand and forms at least one double-stranded region with the first strand. The asdRNA molecules comprise at least one deoxyribonucleotide monomer interspersed segment (ISD), which has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deoxyribonucleotide monomers and can be present in either or both strands. The total number of deoxyribonucleotide monomers in an asdRNA molecule of the invention is less than or equal to the total number of ribonucleotide monomers in any given asdRNA molecule. In one aspect, at least a portion of at least one targeted region in the first strand forms at least one double-stranded region with the second strand.

[0014] The terms "target" and "targeted" are used interchangeably in this disclosure and share the same meaning.

[0015] In one aspect, asdRNAs with an ISD have improved gene modulation or pharmaceutical properties compared to corresponding (asymmetric) RNA duplexes without an ISD. In other words, at least one gene modulation or pharmaceutical property is better or more desirable when the asdRNA comprises at least one ISD disclosed herein; the property is selected from the group consisting of function in various subcellular locations other than the cytoplasm, potential target RNAs of interest, efficacy, potency, off-target effects, speed of initiation, durability, economy of synthesis, accessible chemical modifications, non-specific immune stimulation, stability, and delivery. More specifically, improved gene modulation or pharmaceutical properties of the asdRNA molecules of the present invention compared to corresponding RNA duplexes means, for example, that one or more of the following is true: they can achieve gene modulation function not only in the cytoplasm of cells but also in the nucleus and / or mitochondria; they can target more RNA types (not only mRNA but also pre-mRNA, non-coding RNA, long non-coding RNA, and mt-mRNA (mitochondrial messenger RNA)); they have better efficacy and / or potency, reduced off-target effects, a more rapid onset of action, improved pharmacokinetic properties, longer durability, less dosage-dependent typical toxicity, avoidance of non-specific interferon-like responses, and lower manufacturing costs; they can tolerate / have more chemical modifications (including non-RNA-like nucleotide modifications or substitutions); they have better stability, and they are better delivered. A corresponding (asymmetric) RNA duplex in this application refers to an (asymmetric) short double-stranded RNA molecule that does not have an ISD, and whose antisense strand targets the same or substantially the same sequence as at least one targeted region of the first strand of the asdRNA molecule.More specifically, asdRNAs with an ISD can also be used to target or silence RNAs in the nucleus, such as pre-mRNA, non-coding RNA, and long non-coding RNA, as well as RNAs in mitochondria, such as mt-mRNA (mitochondrial messenger RNA), whereas RNAi technologies with (asymmetric) short double-stranded RNA structures, such as aiRNA and siRNA, function only in the cytoplasm. Therefore, asdRNAs with an ISD can be used to target a larger number of target RNAs, including pathogenic genes, and can be more widely applied than existing gene silencing technologies, particularly RISC-dependent RNAi gene silencing technologies. In another aspect, the asdRNA molecules of the present invention have improved gene modulation or pharmaceutical properties compared to corresponding single-stranded antisense oligonucleotides (ASOs). In other words, the asdRNA has at least one gene modulation or pharmaceutical property that is better or more desirable than the corresponding ASO, and the property is selected from the group consisting of efficacy, potency, speed of onset, durability, economy of synthesis, off-target effects, non-specific immune stimulation, stability, and delivery. The corresponding ASO refers to a single-stranded antisense oligonucleotide that targets the same or substantially the same sequence as at least one targeted region of the first strand of the asdRNA molecule.

[0016] The compositions provided by the present invention are used to modulate gene expression or function in eukaryotic cells, and asdRNA is contacted with the cells or administered to a subject.

[0017] In certain aspects of the asdRNA molecules of the present invention, the first strand of the molecule may contain at least one ISD, and / or the second strand of the molecule may contain at least one ISD. In certain embodiments, the first strand contains at least one ISD, and the second strand also contains at least one ISD. In one aspect, at least one ISD is located within at least one targeting region of the first strand, and at least one ISD is located within at least one double-stranded region of the second strand. In certain embodiments, the first strand contains at least one ISD, but the second strand is composed of ribonucleotide monomers.

[0018] In one aspect, each ISD, independently of the others, consists of one deoxyribonucleotide monomer or comprises at least two, three, four, five, or more consecutive deoxyribonucleotide monomers. In one embodiment, at least one ISD comprises at least four consecutive deoxyribonucleotide monomers. In another aspect, an ISD comprises at least two deoxyribonucleotide monomers, whether consecutive or separated by at least one (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) intervening monomer of a different type. In one aspect, an ISD is separated by at least two (2, 3, 4, 5, 6, 7, 8, 9, 10, or more) intervening monomers of a different type. In yet another aspect, the total number of deoxyribonucleotide monomers of all ISDs in the first strand is at least two.

[0019] In some embodiments, at least one ISD is located within the first strand. In one feature, at least one ISD is located within at least one targeting region of the first strand. In various embodiments, at least one ISD within the targeting region of the first strand comprises at least two, three, four, five, six, seven, eight, nine, or ten consecutive deoxyribonucleotide monomers. In various embodiments, at least one ISD within the first strand comprises at least four consecutive deoxyribonucleotide monomers. In various embodiments, at least one ISD within the targeting region of the first strand comprises at least four consecutive deoxyribonucleotide monomers. In some embodiments, there is only one ISD within the first strand, and the ISD comprises at least four consecutive deoxyribonucleotide monomers. In another embodiment, there are two or more ISDs within the first strand, and each ISD independently consists of one deoxyribonucleotide monomer or comprises at least two, three, four, five, or more consecutive deoxyribonucleotide monomers. In another embodiment, there are two or more ISDs in the first strand, one ISD comprising at least four consecutive deoxyribonucleotide monomers, and the other ISDs each independently consist of one deoxyribonucleotide monomer or comprise at least two, three, four, five or more consecutive deoxyribonucleotide monomers.

[0020] In certain embodiments, at least one ISD is located within the second strand. In one feature, at least one ISD is located within at least one double-stranded region of the second strand. In various embodiments, at least one ISD within the second strand comprises at least two, three, four, five, six, seven, eight, nine, or ten contiguous deoxyribonucleotide monomers. In another feature, the ISD within the second strand comprises at least two deoxyribonucleotide monomers, whether contiguous or separated by at least one (one, two, three, four, five, six, seven, eight, nine, ten, or more) intervening monomer of a different type.

[0021] In some embodiments, the ISD is located more centrally in the first strand (at least 1, 2, 3, 4, or 5 nucleotides away from either end, i.e., starting from position 2 or more centrally when counting from the ends). In some embodiments, the ISD may be located at any position in the second strand. In some embodiments, the ISD is located more centrally in the second strand (at least 1, 2, 3, 4, or 5 nucleotides away from either end, i.e., starting from position 2 or more centrally when counting from the ends). In some embodiments, at least one of the ends of the first strand and / or second strand (i.e., the first nucleotide monomer when counting from the 3' end, 5' end, or both ends) is not a deoxyribonucleotide monomer.

[0022] In one aspect, the first strand comprises a plurality of linked nucleotide monomers forming a nucleobase sequence that is at least 70%, 80%, 85%, 90%, 95%, or fully complementary to a targeted segment of RNA of a target gene. In certain embodiments, the target RNA is selected from mRNA, pre-mRNA, mt-mRNA, and non-coding RNA, which encodes a protein involved in a disease, e.g., a mammalian disease, or controls a portion of a biological pathway involved in a disease, e.g., a mammalian disease.

[0023] In various embodiments, the first strand has a backbone length of 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 linked nucleotide monomers, or an equivalent thereof, or a range between any two of the foregoing values, inclusive of the endpoints of the range. For example, some length ranges for the first antisense strand include: (a) 8 to 33 nucleotide monomers; (b) 10 to 30 nucleotide monomers; (c) 10 to 29 nucleotide monomers; (d) 12 to 29 nucleotide monomers; (e) 12 to 28 nucleotide monomers; (f) 12 to 26 nucleotide monomers; (g) 12 to 25 nucleotide monomers; (h) 13 to 25 nucleotide monomers; (i) 13 to 24 nucleotide monomers; (j) 13 to 23 nucleotide monomers; (k) 15 to 23 nucleotide monomers; (l) 8 to 50 nucleotide monomers; (m) 10 to 36 nucleotide monomers; (n) 12 to 36 nucleotide monomers; (o) 12 to 32 nucleotide monomers; (p) 14 to 36 nucleotide monomers; and (q) at least 8 nucleotide monomers. In some embodiments, when the first strand has a backbone length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 linked nucleotide monomers or equivalent, the at least one ISD may be located at any position in the first strand or, if present in the second strand, at any position in the second strand.

[0024] In one feature, the second strand comprises a plurality of linked nucleotide monomers forming a nucleobase sequence and is at least 70%, 75%, 80%, 85%, 90%, 95%, or fully complementary to at least one linked region of the first strand. In some embodiments, the sense strand is fully complementary to at least one linked region of the first strand, forming at least one double-stranded region without any mismatches. In some embodiments, the sense strand is complementary to at least one linked region of the first / antisense strand, forming at least one double-stranded region with one, two, three, or more mismatches. In one feature, the mismatched monomers in the sense strand have a nucleobase selected from the group consisting of A, G, C, U, and T, or a modified nucleobase. In certain embodiments, at least one of the first base (i.e., the 5'-terminal nucleobase) and the last base (i.e., the 3'-terminal nucleobase) of the second strand is complementary to a nucleobase in the first strand. In some embodiments, at least the first and last bases of the second strand are complementary to nucleobases in the first strand.

[0025] In one aspect, the second strand has a backbone length that is shorter than the first strand by at least the following number of nucleotide monomers: 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, and 38.

[0026] In various embodiments, the second strand has a backbone length of 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, or 36 linked nucleotide monomers, or an equivalent thereof, or a range between any two of the foregoing values, inclusive of the endpoints of the range. In certain embodiments, for example, some of the length ranges of the second sense strand are: (a) 8 to 32 nucleotide monomers; (b) 8 to 30 nucleotide monomers; (c) 8 to 29 nucleotide monomers; (d) 9 to 29 nucleotide monomers; (e) 9 to 26 nucleotide monomers; (f) 9 to 25 nucleotide monomers; (g) 10 to 29 nucleotide monomers; (h) 10 to 28 nucleotide monomers; (i) 10 to 26 nucleotide monomers; (j) 10 to 25 nucleotide monomers; (k) 11 to 24 nucleotide monomers; (l) 11 to 23 nucleotide monomers; (m) 12 to 23 nucleotide monomers; (n) 12 to 22 nucleotide monomers; (o) a tie of 13-23 nucleotide monomers; (p) a tie of 15-23 nucleotide monomers; (q) a tie of 8-35 nucleotide monomers; (r) a tie of 8-33 nucleotide monomers; (s) a tie of 9-35 nucleotide monomers; (t) a tie of 9-34 nucleotide monomers; (u) a tie of 9-32 nucleotide monomers; (v) a tie of 9-30 nucleotide monomers; (w) a tie of 10-30 nucleotide monomers; (x) a tie of 10-32 nucleotide monomers; (y) at least 8 nucleotide monomers, and (z) at least 6 nucleotide monomers. In certain embodiments, the second strand can have a backbone length of any number of nucleotide monomers less than that of the first strand, provided that it is thermodynamically capable of forming a duplex with the first strand.

[0027] In one aspect, the two ends of the first strand are in one of the following configurations: a 3'-overhang and a 5'-overhang; a 3'-overhang and a blunt end at the 5'-end; a 5'-overhang and a blunt end at the 3'-end; a 3'-overhang and a 5' recessed end; or a 5'-overhang and a 3' recessed end. In certain embodiments, the 3'-overhang of the first strand has a length 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, or 30 nucleotide monomers, or a range defined between any two of the foregoing values, inclusive of the endpoints of the range. In various embodiments, the 3'-overhang of the first strand has a length of 1 to 15, 1 to 10, 1 to 9, 1 to 8, or 1 to 5 nucleotide monomers (inclusive of both end points of the ranges). In certain embodiments, the 5'-overhang of the first strand has a length 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, or 30 nucleotide monomers, or a range defined between any two of the foregoing values, inclusive of the endpoints of the ranges. In various embodiments, the 5'-overhang of the first strand has a length of 1 to 15, 1 to 10, 1 to 9, 1 to 8, or 1 to 5 nucleotide monomers, inclusive of the endpoints of the ranges.

[0028] In one embodiment of the invention, the first strand has a 3'-overhang of 1 to 15 nucleotide monomers and a 5'-overhang of 1 to 15 nucleotide monomers. In another embodiment, the first strand has a 3'-overhang of 1 to 26 nucleotide monomers and a 5' blunt end or a 5' recessed end. In yet another embodiment, the first strand has a 5'-overhang of 1 to 26 nucleotide monomers and a 3' blunt end or a 3' recessed end.

[0029] In one aspect, the two ends of the second strand are in one of the following configurations: a 3'-overhang and a 5'-recessed end; a 5'-overhang and a 3'-recessed end; a 3'-blunt end and a 5'-recessed end; a 5'-blunt end and a 3'-recessed end; a 3'-recessed end and a 5'-recessed end. In certain embodiments, the 3'-overhang of the second strand has a length 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, or 25 nucleotide monomers. In various embodiments, the 3'-overhang of the second strand has a length of 1 to 15, 1 to 10, 1 to 9, 1 to 8, or 1 to 5 nucleotide monomers (inclusive of both end points of the ranges). In certain embodiments, the 5'-overhang of the second strand has a length 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, or 25 nucleotide monomers. In various embodiments, the 5'-overhang of the second strand has a length of 1 to 15, 1 to 10, 1 to 9, 1 to 8, or 1 to 5 nucleotide monomers (inclusive of both end points of the ranges).

[0030] In some aspects of the asdRNA molecules of the present invention, at least one nucleotide monomer in the first and / or second strand is a modified nucleotide or nucleotide analog, such as a sugar-modified nucleotide, a backbone-modified nucleotide, and / or a base-modified nucleotide. In some embodiments, such backbone-modified nucleotides have at least one internucleoside linkage modified, for example, to include at least one nitrogen or sulfur heteroatom. In some embodiments, the modified internucleoside linkage is or includes a phosphorothioate (P=S) group, a phosphotriester, a methylphosphonate, or a phosphoramidate.

[0031] In certain embodiments, the first and / or second strands comprise at least one modified internucleoside linkage, and the modified internucleoside linkage is a phosphorothioate internucleoside linkage. In some embodiments, each internucleoside linkage in the first and / or second strands is a phosphorothioate internucleoside linkage. In various embodiments, the internucleoside linkages in the first and / or second strands are a mixture of phosphorothioate and phosphodiester linkages. In some embodiments, each internucleoside linkage in the first strand is a modified internucleoside linkage, while each internucleoside linkage in the second strand is a naturally occurring internucleoside linkage.

[0032] In one aspect, the first and / or second strand of a molecule of the invention comprises at least one modified nucleotide or nucleotide analog comprising a modified sugar moiety. In certain embodiments, the 2'-position of the modified sugar moiety is replaced with a group selected from OR, R, halo, SH, SR, NH, NHR, NR, or CN, where each R is independently C-C alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I. In some embodiments, the 2'-position of the modified sugar moiety is selected from allyl, amino, azido, thio, O-allyl, O-C-C 10 Alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), O-CH2-C(=O)-N(R m )(R n ), or O-CH2-C(=O)-N(R1)-(CH2)2-N(R m )(R n ), wherein each R l , R m and R n are independently H or substituted or unsubstituted C1-C 10In some embodiments, the modified sugar moiety has a substituent selected from the group of 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH, 2'-OCHCH, 2'-OCHCHF, 2'-O-aminopropylated (2'-AP), and 2'-O(CH)OCH. In some embodiments, the modified sugar moiety is substituted with a bicyclic sugar selected from the group of 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (cEt), and 4'-CH(CHOCH3)-O-2', 4'-C(CH3)(CH3)-O-2', 4'-CH2-N(OCH3)-2', 4'-CH2-ON(CH3)-2', 4'-CH2-N(R)-O-2' (where R is H, C1-C12 alkyl, or a protecting group), 4'-CH2-C(H)(CH3)-2', and 4'-CH2-C-(=CH2)-2'. In some embodiments, the modified sugar moiety is selected from the group of 2'-O-methoxyethyl modified sugars (MOE), 4'-(CH2)-O-2' bicyclic sugars (LNA), 2'-deoxy-2'-fluoroarabinose (2'-F-arabino, FANA), and methyl(methyleneoxy) (4'-CH(CH3)-O-2) bicyclic sugars (cEt).

[0033] In certain embodiments, the ISD comprises at least one modified nucleotide or nucleotide analog having a modified sugar moiety, wherein the modified sugar moiety is 2'-deoxy-2'-fluoroarabinose (FANA).

[0034] In yet another particular embodiment, the ISD may comprise at least one CpG motif that can be recognized by a pattern recognition receptor (PRR), such as a Toll-like receptor.

[0035] In one aspect of the asdRNA molecules of the invention, the sugar moieties of the ribonucleotide monomers are selected from naturally occurring ribonucleotides (2-OH), 2'-F modified sugars, 2'-OMe modified sugars, 2'-O-methoxyethyl modified sugars (MOE), 4'-(CH2)-O-2' bicyclic sugars (LNA), and methyl(methyleneoxy) (4'-CH(CH3)-O-2) bicyclic sugars (cEt).

[0036] In one aspect of the asdRNA molecules of the invention, the sugar moiety of the deoxyribonucleotide monomer is either the sugar moiety of a naturally occurring deoxyribonucleotide (2-H) or 2'-deoxy-2'-fluoroarabinose (FANA).

[0037] In another aspect, the first and / or second strand of the molecules of the invention comprise at least one nucleotide monomer comprising a modified nucleobase. In some embodiments, the modified nucleobase is 5-methylcytosine (5-Me-C), inosine bases, tritylated bases, 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-propynyl (-C≡C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases; 6-azouracil, cytosine and thymine, 5-uracil The modified nucleic acid base is selected from the group consisting of 1-methyl-pseudouracil, 4-thiouracil, 1-methyl-pseudouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-methyluridine and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.In certain embodiments, the modified nucleic acid base is 5-methylcytosine.In some embodiments, each cytosine base in the molecule of the present invention is 5-methylcytosine. In certain embodiments, each uridine base in a ribonucleotide monomer of an asdRNA molecule of the present invention is 5-methyluridine.

[0038] In one aspect, the first and / or second strands of the molecules of the invention are conjugated to a ligand or moiety. In certain embodiments, the ligand or moiety is selected from the group consisting of a peptide, an antibody, a polymer, a polysaccharide, a lipid, a hydrophobic moiety or molecule, a cationic moiety or molecule, a lipophilic compound or moiety, an oligonucleotide, cholesterol, GalNAc, and an aptamer.

[0039] In one aspect of the invention, asdRNA molecules are used to modulate gene expression or function in cells, eg, eukaryotic cells, such as mammalian cells.

[0040] In certain embodiments, the target RNA that directs at least a portion of the nucleotide monomer sequence of an asdRNA molecule according to the principles of the present invention is selected from mRNA, pre-mRNA, mt-mRNA, or non-coding RNA. In one aspect, such target RNA encodes a protein involved in a disease or regulates a portion of a biological pathway involved in a disease. In various embodiments, such target RNA may be selected from, but is not limited to, mRNA, pre-mRNA, mt-mRNA, non-coding RNA, or lncRNA of a gene involved in a human or animal disease or condition; mRNA or pre-mRNA of a gene of a pathogenic microorganism; viral RNA, and RNA involved in a disease or disorder selected from the group consisting of autoimmune diseases, inflammatory diseases, degenerative diseases, infectious diseases, proliferative diseases, metabolic diseases, immune-mediated disorders, allergic diseases, dermatological diseases, malignant diseases, gastrointestinal disorders, respiratory disorders, cardiovascular disorders, nephropathy, rheumatoid disorders, neurological disorders, endocrine disorders, and aging-related disorders or diseases.

[0041] In one embodiment, the present invention provides an asymmetric short duplex RNA (asdRNA) molecule comprising a first strand and a second strand, each strand comprising linked ribonucleotide monomers selected from the group consisting of naturally occurring ribonucleotides, their analogs, and modified ribonucleotides, and at least one deoxyribonucleotide monomer interspersed segment (ISD), wherein: (a) the first strand is longer than the second strand by a number of monomers selected from the group consisting of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 monomers; (b) the first strand is substantially complementary to a segment of an RNA targeted by at least one targeting region, wherein the first strand consists of 10 to 36 (inclusive) nucleoside monomers linked between adjacent monomers by linkages selected from the group consisting of phosphorothioate linkages, phosphodiester linkages, and mixtures of phosphorothioate and phosphodiester linkages; and (c) the second strand is substantially complementary to a segment of an RNA targeted by at least one targeting region, wherein the first strand consists of 10 to 36 (inclusive) nucleoside monomers linked between adjacent monomers by linkages selected from the group consisting of phosphorothioate linkages, phosphodiester linkages, and mixtures of phosphorothioate and phosphodiester linkages; (d) at least one ISD linked to at least one ribonucleotide monomer selected from the group consisting of ribonucleotides, their analogs, and modified ribonucleotides; (e) the ISD in the asdRNA molecule comprises at least one deoxyribonucleotide monomer selected from the group consisting of deoxyribonucleotides, their analogs, and modified deoxyribonucleotides; and (f) the total number of deoxyribonucleotide monomers is equal to or less than the total number of ribonucleotide monomers in the asdRNA molecule. In one aspect, the asdRNA molecule is used to modulate target gene expression or function in a cell, e.g., a eukaryotic cell, e.g., a mammalian cell. In a further aspect, the asdRNA molecule is more potent or effective at silencing expression of a target gene in a cell than the corresponding ASO.In a further aspect, asdRNA molecules can achieve their gene modulation function not only in the cytoplasm but also in the nucleus and / or mitochondria of cells, and thus can target RNA in the nucleus and / or mitochondria of cells. In a further aspect, asdRNA molecules are more potent or effective at silencing target gene expression in cells than corresponding (asymmetric) RNA duplexes. The mechanism of asdRNA in the present invention is unclear, but currently, studies have shown that the gene silencing efficacy of asdRNA in the present invention is not affected when Ago2 is knocked down, indicating that asdRNA in the present invention may not function via a RISC-dependent mechanism. Therefore, asdRNA can be used to target genes or target sequences of interest that are refractory or insensitive to siRNA or asymmetric siRNA (aiRNA).

[0042] In a second aspect, the present invention provides a pharmaceutical composition comprising the composition of the first aspect as an active agent and a pharmaceutically acceptable excipient, carrier or diluent.Examples of such carriers include, but are not limited to, pharmaceutical carriers, positively charged carriers, liposomes, lipid nanoparticles, protein carriers, hydrophobic moieties or molecules, cationic moieties or molecules, GalNAc, polysaccharides, polymers, nanoparticles, nanoemulsions, cholesterol, lipids, lipophilic compounds or moieties, and lipids.

[0043] In a third aspect, the present invention provides a method of using the composition of the first aspect or the pharmaceutical composition of the second aspect to treat or prevent a disease or condition by administering a therapeutically effective amount of an asdRNA molecule of the invention or a pharmaceutical composition containing such a molecule by a route selected from the group consisting of intravenous injection (iv), subcutaneous injection (sc), orally (po), intramuscular (im) injection, oral administration, inhalation, topical, intrathecal, and other localized administration.

[0044] In one aspect, the disease or condition to be treated prophylactically or therapeutically is selected from the group of cancer, autoimmune diseases, inflammatory diseases, degenerative diseases, infectious diseases, proliferative diseases, metabolic diseases, immune-mediated disorders, allergic diseases, dermatological diseases, malignant diseases, gastrointestinal disorders, hepatic disorders, respiratory disorders, cardiovascular disorders, dermatological disorders, nephrological disorders, rheumatoid disorders, neurological disorders, psychiatric disorders, endocrine disorders, and aging-related disorders or diseases.

[0045] In a fourth aspect, the present invention provides a method of using the composition of the first aspect or the pharmaceutical composition of the second aspect to control or modulate gene expression or function in a eukaryotic cell, the method comprising contacting the cell with an effective amount of any asdRNA molecule of the present invention or a pharmaceutical composition containing such molecule.

[0046] In one embodiment, the contacting step comprises introducing a composition comprising the asdRNA molecule into target cells in culture or into target cells in an organism capable of selective gene silencing. In a further embodiment, the introducing step is selected from the group consisting of simple mixing, transfection, lipofection, electroporation, infection, injection, and oral administration, intravenous injection (iv), subcutaneous injection (sc), oral (po), intramuscular (im) injection, inhalation, topical, intrathecal, and other localized administration. In another embodiment, the introducing step comprises using a pharmaceutically acceptable excipient, carrier, or diluent selected from the group consisting of a pharmaceutical carrier, a positively charged carrier, a lipid nanoparticle, a liposome, a protein carrier, a hydrophobic moiety or molecule, a cationic moiety or molecule, GalNAc, a polysaccharide, a polymer, a nanoparticle, a nanoemulsion, cholesterol, a lipid, a lipophilic compound or moiety, and a lipid.

[0047] In certain embodiments, the target RNA is mRNA. In certain embodiments, the target RNA is pre-mRNA. In certain embodiments, the target RNA is mt-mRNA. In certain embodiments, the target RNA is non-coding RNA, such as microRNA and lncRNA.

[0048] In one embodiment, the target gene is associated with a disease, pathological condition, or undesirable condition in a mammal. In a further embodiment, the target gene is a gene of a pathogenic microorganism. In yet a further embodiment, the target gene is a viral gene. In another embodiment, the target gene is a tumor-associated gene. In yet another embodiment, the target gene is a gene associated with a disease selected from the group listed for the third aspect.

[0049] In another aspect, the present invention provides an asymmetric oligomeric duplex comprising (a) one or more ribonucleosides, analogs thereof, or modified ribonucleosides, and (b) one or more ISDs comprising deoxyribonucleosides, analogs thereof, or modified deoxyribonucleosides linked within an antisense sequence, and thus having a length of at least 8 nucleobases, wherein the antisense sequence is at least 70% complementary to a target sequence.

[0050] Other features and advantages of the present invention will be apparent from the additional description provided herein, including the different examples. The provided examples illustrate different components and methodologies useful in practicing the present invention. The examples do not limit the claimed invention. Based on this disclosure, one skilled in the art will be able to identify and utilize other components and methodologies useful in practicing the present invention. While several embodiments have been shown and described, any modifications can be made without departing from the spirit and scope of the present invention. [Brief explanation of the drawings]

[0051] [Figure 1]FIG. 1 shows representative target genes and representative target sequences used in the examples, as well as exemplary sequences of the corresponding antisense strands of molecules that can be used to silence the target genes.

[0052] [Figure 2-1] Figure 2A shows exemplary structures of some embodiments of asdRNAs with at least one deoxyribonucleotide monomer interspersed segment (ISD) in the antisense strand (first strand) and the pure RNA sense strand (second strand). For each duplex depicted here, the sense strand is listed above the antisense strand. Figure 2B shows exemplary sequences of asdRNAs with the structures in Figure 2A that target the APOCIII gene. Figure 2C shows the gene silencing efficacy of asdRNAs with the sequences in Figure 2B that target the APOCIII gene, compared with the corresponding ASOs (the corresponding ASOs have the same sequence as the antisense strand of each asdRNA in Figure 2B). After transfection of the asdRNAs and the corresponding ASOs at 100 pM into HepaRG cells, the relative mRNA levels of the APOCIII gene were determined. [Figure 2-2] Same as above. [Figure 2-3] Same as above.

[0053] [Figure 3-1]Figure 3A shows exemplary structures of some embodiments of asdRNAs with various ISD motifs in the antisense strand, as well as exemplary sequences of asdRNAs targeting the APOCIII gene. The various ISD motifs in the antisense strand in Figure 3A have various numbers of deoxyribonucleotide monomers and positions of the ISD in the antisense strand. Figure 3B shows a comparison of the gene silencing efficacy of the asdRNAs targeting the APOCIII gene shown in Figure 3A with that of the corresponding ASOs (each of which has the same sequence as the antisense strand of each asdRNA in Figure 3A). After transfection of the asdRNAs and the corresponding ASOs at 100 pM into HepaRG cells, the relative mRNA levels of the APOCIII gene were determined. [Figure 3-2] Same as above.

[0054] [Figure 4-1] Figure 4A shows the exemplary structure of some embodiments of asdRNA with at least one ISD only on the antisense strand, and the exemplary sequence of asdRNA targeting APOB gene. Figure 4B shows the gene silencing efficacy of asdRNA with the structure in Figure 4A targeting APOB gene. After asdRNA was introduced into HepaRG cells by transfection at 5 nM, the relative mRNA level of APOB gene was determined. [Figure 4-2] Same as above.

[0055] [Figure 5] FIG. 5 shows the sequences and gene silencing potencies in DLD1 cells at 100 pM, 200 pM, 1 nM, 3 nM, 10 nM, and 30 nM, respectively, for exemplary asdRNAs targeting the β-catenin gene. DETAILED DESCRIPTION OF THE INVENTION

[0056] Detailed Description of the Invention The present invention refers to a gene or RNA modulation / silencing technology that uses a novel type of short double-stranded RNA. This new technology is used to modulate gene expression or function in vitro and in vivo by using asymmetric short double-stranded RNA (asdRNA) compositions with deoxynucleotide-interspersed segments. The present invention also provides methods for using the compositions to modulate the expression or function of target genes, or for the treatment or prevention of diseases, as well as for other medical and biological applications. These compositions and methods provide high efficacy in controlling gene expression or gene function, while also reducing dose-dependent toxicity.

[0057] 1.Definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0058] When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below those numerical values. Generally, the term "about" is used herein to modify a numerical value by a variance of 20%, 10%, 5%, or 1% above and below the stated value. In some embodiments, the term "about" is used to modify a numerical value by a variance of 10% above and below the stated value. In some embodiments, the term "about" is used to modify a numerical value by a variance of 5% above and below the stated value. In some embodiments, the term "about" is used to modify a numerical value by a variance of 1% above and below the stated value.

[0059] As used herein, the term " analog " or " analogue " refers to a functional or structural equivalent.For example, nucleoside and nucleotide analogues have been used in the clinical treatment of cancer and viral infection for decades, and new compounds are continuously synthesized and evaluated by researchers and the pharmaceutical industry.See, for example, Jordheim LP et al., Nat Rev Drug Discov 12, 447-464 (2013).

[0060] As used herein, the term "deoxyribonucleoside monomer" refers to a nucleoside monomer, including naturally occurring deoxyribonucleosides, their analogs, and modified deoxyribonucleosides. The term "deoxyribonucleotide monomer" refers to a nucleotide monomer, including naturally occurring deoxyribonucleotides, their analogs, and modified deoxyribonucleotides.

[0061] As used herein, the term "ribonucleoside monomer" refers to a nucleoside monomer, including naturally occurring ribonucleosides, their analogs, and modified ribonucleosides. The term "ribonucleotide monomer" refers to a nucleotide monomer, including naturally occurring ribonucleotides, their analogs, and modified ribonucleotides.

[0062] As used herein, the term "nucleoside" refers to a compound comprising a nucleobase moiety and a sugar moiety. Nucleoside monomers include, but are not limited to, naturally occurring nucleosides (e.g., deoxyribonucleosides and ribonucleosides found in DNA and RNA, respectively), their analogs, and modified nucleosides. Nucleoside monomers can be deoxyribonucleoside monomers or ribonucleoside monomers. Nucleoside monomers can be linked to a phosphate moiety, for example, to form nucleotide monomers.

[0063] As used herein, the term "nucleotide" refers to a nucleoside that further comprises a phosphate linking group. Nucleotide monomers include, but are not limited to, naturally occurring nucleotides (e.g., deoxyribonucleotides and ribonucleotides, as found in DNA and RNA, respectively), their analogs, and modified nucleotides. A nucleotide monomer may be a deoxyribonucleotide monomer or a ribonucleotide monomer. A modified nucleotide may be modified in one or more of the following: its nitrogenous nucleobase moiety, its five-carbon sugar moiety, and its phosphate linking group, resulting in a change in the internucleoside linkage.

[0064] As used herein, the term "oligo" or "oligonucleotide" refers to a compound comprising multiple linked nucleoside monomers. In certain embodiments, one or more of the nucleoside monomers or one or more of the internucleoside linkages are modified.

[0065] The terms "deoxynucleoside" and "deoxyribonucleoside" are used interchangeably herein. The terms "deoxynucleotide" and "deoxyribonucleotide" are also used interchangeably herein. As used herein, a "deoxynucleoside" or "deoxynucleotide" is a nucleoside or nucleotide, respectively, that contains a deoxy sugar moiety.

[0066] As used herein, the term "double-stranded RNA," as in "short double-stranded RNA (sdRNA)" or "asymmetric short double-stranded RNA (asdRNA)," refers to a molecule composed of two strands or chains of nucleotide monomers that hybridize to each other to form a double-stranded oligonucleotide, contacted with a cell, or administered to a subject, wherein the majority of the linked nucleotide monomers, i.e., 50% or more, are ribonucleotide monomers that contain modified ribonucleotides.

[0067] As used herein, the term "motif" refers to a pattern of chemically distinct regions, for example, within an antisense or sense strand.

[0068] As used herein, the term "immediately adjacent" means that there are no intervening elements between the two elements, eg, regions, segments, nucleotides and / or nucleosides.

[0069] As used herein, the term "modified nucleotide" means a nucleotide having at least one modified sugar moiety, modified internucleoside linkage, and / or modified nucleobase.

[0070] As used herein, the term "modified nucleoside" means a nucleoside having at least one modified sugar moiety and / or modified nucleobase.

[0071] As used herein, the term "modified oligonucleotide" means an oligonucleotide that contains at least one modified nucleotide.

[0072] As used herein, the term "naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage.

[0073] As used herein, the term "modified internucleoside linkage" refers to a substitution or any change from a naturally occurring internucleoside linkage. For example, a phosphorothioate linkage is a modified internucleoside linkage.

[0074] As used herein, the term "natural sugar moiety" means a sugar that is naturally found in DNA (2-H) or RNA (2-OH).

[0075] As used herein, the term "modified sugar" refers to a substitution or change from a natural sugar. For example, a 2'-O-methoxyethyl modified sugar is a modified sugar.

[0076] As used herein, the term "bicyclic sugar" means a furosyl ring modified by bridging two non-geminal ring atoms. A bicyclic sugar is a modified sugar.

[0077] As used herein, the term "bicyclic nucleic acid," "BNA," "bicyclic nucleoside," or "bicyclic nucleotide" refers to a nucleoside or nucleotide in which the furanose portion of the nucleoside or nucleotide includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system.

[0078] As used herein, the term "2'-O-methoxyethyl" (also 2'-MOE, 2'-O(CH2)2-OCH3, and 2'-O-(2-methoxyethyl)) refers to an O-methoxy-ethyl modification at the 2' position of the furosyl ring. A 2'-O-methoxyethyl modified sugar is a modified sugar. As used herein, the term "2'-O-methoxyethyl nucleotide" (also 2'-MOE RNA) refers to a modified nucleotide containing a 2'-O-methoxyethyl modified sugar moiety.

[0079] As used herein, the term "modified nucleobase" refers to any nucleobase other than adenine, cytosine, guanine, thymidine or uracil.For example, 5-methylcytosine is a modified nucleobase.On the other hand, as used herein, "unmodified nucleobase" refers to purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C) and uracil (U).

[0080] As used herein, the term "5-methylcytosine" means a cytosine modified with a methyl group attached to position 5. For example, 5-methylcytosine is a modified nucleobase.

[0081] As used herein, "RNA-like nucleotides" refers to modified nucleotides that, when incorporated into an oligonucleotide, assume a Northern configuration and function like RNA. RNA-like nucleotides include, but are not limited to, bridged nucleic acids (BNA), LNA, cEt, 2'-O-methylated nucleotides, 2'-O-methoxyethylated (2'-MOE) nucleotides, 2'-fluorinated nucleotides, 2'-O-aminopropylated (2'-AP) nucleotides, tricyclo-DNA (tcDNA), and RNA surrogates.

[0082] As used herein, "DNA-like nucleotides" refers to modified nucleotides that function like DNA when incorporated into an oligonucleotide. DNA-like nucleotides include, but are not limited to, 2'-deoxy-2'-fluoroarabinose (FANA) nucleotides and DNA surrogates.

[0083] As used herein, "non-coding RNA" refers to an RNA molecule that is not translated into protein. Examples of non-coding RNA include transfer RNA (tRNA) and ribosomal RNA (rRNA), as well as small non-coding RNA and long ncRNA (lncRNA). As used herein, examples of "small non-coding RNA" include, but are not limited to, microRNA (miRNA), asRNA, pre-miRNA, pri-miRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, and mimics of any of the foregoing. As used herein, "lncRNA" and "long non-coding RNA" refer to transcribed RNA molecules containing more than 200 nucleotides that do not encode proteins. lncRNAs can also undergo common post-transcriptional modifications, including 5'-capping, 3'-polyadenylation, and splicing. In general, IncRNAs are a diverse class of molecules that play various roles in modifying gene and genome function. For example, it is known that lncRNAs control gene transcription, translation, and epigenetic regulation. Examples of IncRNAs include, but are not limited to, Kcnqlotl, Xlsirt, Xist, ANRIL, NEAT1, NRON, DANCR, OIP5-AS1, TUG1, CasC7, HOTAIR, and MALAT1. As used herein, "splice" or "splicing" refers to the natural process of removing unnecessary regions of RNA and reforming the RNA. An example of modulation of RNA target function by the asdRNA is modulation of non-coding RNA function. In some embodiments, the asdRNA is designed to target one of the above-mentioned small non-coding RNAs. In some embodiments, the asdRNA is designed to target an miRNA. In some embodiments, the asdRNA is designed to target a pre-miRNA. In some embodiments, the asdRNA is designed to target a pri-miRNA. In some embodiments, the asdRNA is designed to target an lncRNA.In some embodiments, the asdRNA is designed to target a splice.

[0084] Nuclear-targeted RNAs refer to RNA molecules that are synthesized and / or function in the nucleus of a cell. According to preferred embodiments, nuclear-targeted RNAs of the present invention include lncRNAs, non-coding RNAs, pre-mRNAs, and pre-miRNAs. As used herein, the term "pre-mRNA" refers to unprocessed or partially processed precursor mRNAs containing introns and exons, which are synthesized by transcription from a cellular DNA template. Pre-mRNAs require intron splicing (removal) to produce mRNA molecules containing only exons. In some embodiments, asdRNAs are designed to target pre-mRNAs. The terms "mitochondrial messenger RNA" and "mt-mRNA" refer to mRNA molecules transcribed from mitochondrial DNA. In some embodiments, asdRNAs are designed to target mt-mRNAs in mitochondria.

[0085] The terms "isolated" or "purified," as used herein, refer to a material that is substantially or essentially free from components with which it is normally associated in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

[0086] The term "interspersed," as used herein, refers to having portions of different types adjacently spaced apart, e.g., by different types of nucleotides or nucleotide analogs, or different modifications to the same type of nucleotide or nucleotide analog. In various embodiments of the present invention, a "deoxyribonucleotide monomer interspersed segment (ISD)" refers to a section of an oligonucleotide strand in which one or more deoxyribonucleotides are connected to at least one portion that is a different type from the deoxyribonucleotides. For example, if the deoxyribonucleotides are unmodified, the portions of different types can be ribonucleotides or analogs thereof, modified ribonucleotides, modified deoxyribonucleotides, or deoxyribonucleotide analogs. If the deoxyribonucleotides are modified, the portions of different types can be ribonucleotides or analogs thereof, modified ribonucleotides, unmodified deoxyribonucleotides, differently modified deoxyribonucleotides, or different types of deoxyribonucleotide analogs.

[0087] As used herein, " modulating," "regulating," and its grammatical equivalents refer to either increasing or decreasing (e.g., silencing), in other words, either up-regulating or down-regulating.As used herein, "gene silencing" refers to the reduction of gene expression, and can refer to the reduction of gene expression by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the target gene.

[0088] As used herein, the terms "inhibiting," "to inhibit," and their grammatical equivalents, when used in the context of a biological activity, refer to the downregulation of a biological activity, which may reduce or eliminate a targeted function, such as the production of a protein or the phosphorylation of a molecule. In certain embodiments, inhibition may refer to a reduction of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of a targeted activity. When used in the context of a disorder or disease, these terms refer to success in preventing the onset of symptoms, alleviating symptoms, or eliminating a disease, condition, or disorder.

[0089] As used herein, the term "substantially complementary" or "complementary" refers to complementarity in the base-paired double-stranded region between two strands of linked nucleosides, without any single-stranded regions, such as terminal overhangs, or gap regions between the two double-stranded regions. Complementarity does not need to be perfect; for example, there can be any number of base pair mismatches between the two strands of linked nucleosides. However, if the number of mismatches is so great that hybridization does not occur even under minimally stringent hybridization conditions, the sequence is not a substantially complementary sequence. Specifically, when two sequences are referred to herein as "substantially complementary," it means that the sequences are sufficiently complementary to each other to hybridize under the selected reaction conditions. The relationship between nucleic acid complementarity and the stringency of hybridization sufficient to achieve specificity is well known in the art. Two substantially complementary strands can be, for example, perfectly complementary, or can contain one to many mismatches, provided that the hybridization conditions are sufficient to allow, for example, discrimination between matched and unmatched sequences. Thus, substantially complementary sequences can refer to sequences having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% base pair complementarity in the double-stranded region, or any number in between.

[0090] As used herein, "fully complementary" or "100% complementary" means that each nucleobase of the nucleobase sequence of a first strand of linked nucleosides has a complementary nucleobase in the second nucleobase sequence of a second strand of linked nucleosides. In certain embodiments, the first strand of linked nucleosides is an antisense compound, and the second strand of linked nucleosides is a target nucleic acid. In certain embodiments, the first strand of linked nucleosides is a sense compound, and the second strand of linked nucleosides is an antisense compound, or conversely, the first strand of linked nucleosides is an antisense compound, and the second strand of linked nucleosides is a sense compound.

[0091] As used herein, term " targeting region " refers to the region in an oligonucleotide chain, and is substantially or completely complementary to another oligonucleotide chain, and therefore these two chains hybridize or anneal with each other in this targeting region under suitable conditions.For example, antisense chain can comprise targeting region, and can thereby hybridize with target mRNA.

[0092] The terms "administer," "administering," or "administration" are used herein in their broadest sense. These terms refer to any method of introducing a compound or pharmaceutical composition described herein into a subject, for example, administering a compound to a subject systemically, topically, or intravenously. This may include introducing the compound or composition in situ. Thus, compounds of the present disclosure produced in a subject from a composition (whether or not the composition contains the compound) are encompassed by these terms. When these terms are used in conjunction with the terms "systemic" or "systemically," they generally refer to in vivo systemic absorption or accumulation of the compound or composition in the bloodstream, followed by distribution throughout the body.

[0093] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or pharmaceutical compound described herein that is sufficient to affect an intended outcome, including, but not limited to, disease treatment, as described below. In some embodiments, a "therapeutically effective amount" is an amount that is effective in detectably killing or inhibiting the growth or spread of cancer cells, tumor size or number, and / or other measures of the level, stage, progression, and / or severity of cancer. In some embodiments, a "therapeutically effective amount" refers to an amount administered systemically, locally, or in situ (e.g., the amount of compound produced in situ in a subject). A therapeutically effective amount may vary depending on the intended application (in vitro or in vivo), or the subject and condition to be treated, such as the subject's weight and age, the severity of the condition, or the mode of administration, and can be readily determined by one of ordinary skill in the art. The term also applies to a dose that will induce a specific response in target cells, such as reduced cell migration. The specific dose may vary depending, for example, on the particular pharmaceutical composition, the subject and their age and pre-existing health conditions or risk of health conditions, the dosing regimen to be followed, the severity of the disease, whether it is administered in combination with other agents, the timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

[0094] The term "cancer" in a subject refers to the presence of cells that have properties typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain morphological characteristics. Often, cancer cells will exist in the form of a tumor or mass, but such cells may exist alone within a subject or circulate in the bloodstream as independent cells, such as leukemia or lymphoma cells. Examples of cancer as used herein include, but are not limited to, lung cancer, pancreatic cancer, bone cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, breast cancer, uterine cancer, ovarian cancer, peritoneal cancer, colon cancer, rectal cancer, colorectal adenocarcinoma, anal cancer, stomach cancer, gastric cancer, gastrointestinal cancer, gastric adenocarcinoma, adrenocorticoid cancer, and the like. carcinoma), uterine cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, gastroesophageal junction cancer, gastroesophageal adenocarcinoma, chondrosarcoma, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, Ewing's sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, testicular cancer, ureteral cancer, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, biliary tract cancer, kidney cancer, renal cell carcinoma, chronic or acute leukemia, lymphocytic lymphoma, neoplasms of the central nervous system (CNS), spinal axis tumor, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, including refractory versions of any of the foregoing cancers or combinations of one or more of the foregoing cancers. Some of the exemplified cancers are encompassed by the general term and are included in this term. For example, the general term urinary cancer includes bladder cancer, prostate cancer, kidney cancer, testicular cancer, etc., and another general term, hepatobiliary cancer, includes liver cancer (which itself is a general term including hepatocellular carcinoma or biliary tract cancer), gallbladder cancer, biliary tract cancer, or pancreatic cancer. Both urinary cancer and hepatobiliary cancer are contemplated by the present disclosure and are included in the term "cancer."

[0095] The term "pharmaceutical composition" refers to a formulation containing an active ingredient, such as a molecule or composition disclosed herein, in a form suitable for administration to a subject, often in admixture with other substances, e.g., a pharmaceutical carrier such as a sterile aqueous solution. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form may be in any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial. The amount of active ingredient in a unit dose of the composition is an effective amount and will vary according to the particular treatment involved. Those skilled in the art will understand that routine variations in dosage may sometimes be necessary depending on the age and condition of the patient. Dosage will also depend on the route of administration. Various routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, and intranasal routes. Dosage forms for topical or transdermal administration of the asdRNA of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants.

[0096] The term "pharmaceutical agent" means a substance that provides a therapeutic benefit when administered to an individual.

[0097] The term " pharmaceutically acceptable carrier " refers to a medium or diluent that does not interfere with the structure of the compound. Some of these carriers allow pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and lozenges for oral ingestion by subjects. Some of these carriers allow pharmaceutical compositions to be formulated for injection, infusion or local administration. For example, a pharmaceutically acceptable carrier is a sterile aqueous solution.

[0098] The term "pharmaceutically acceptable derivatives" includes derivatives of the compounds described herein, such as solvates, hydrates, esters, prodrugs, polymorphs, isomers, isotopically labeled variants, pharmaceutically acceptable salts, and other derivatives known in the art.

[0099] The term "pharmaceutically acceptable salt" refers to a physiologically and pharmaceutically acceptable salt of a compound, i.e., a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects thereto. The term "pharmaceutically acceptable salt" or "salt" includes salts prepared by reacting the parent compound with a pharmaceutically acceptable non-toxic acid or base, including inorganic or organic acids and bases. Pharmaceutically acceptable salts of the compounds described herein can be prepared by methods well known in the art. For a review of pharmaceutically acceptable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use (Wiley-VCH, Weinheim, Germany, 2002). Pharmaceutically acceptable salts can include, but are not limited to, acid addition salts, including hydrochloride, hydrobromide, phosphate, sulfate, hydrogen sulfate, alkylsulfonate, arylsulfonate, acetate, benzoate, citrate, maleate, fumarate, succinate, lactate and tartrate; alkali metal cations, for example, Na, K, Li, alkaline earth metal salts, for example, Mg or Ca, or organic amine salts.In particular, the sodium salt of oligonucleotide has been proven to be useful and is well tolerated for therapeutic administration to humans.Therefore, in one embodiment, the compound described herein is in the form of sodium salt.

[0100] As used herein, the term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., who will be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0101] The terms "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate," as used herein, refer to both (1) therapeutic measures that cure, slow, reduce the symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent or slow the onset of the targeted pathological condition or disorder. Thus, those in need of treatment include those who already have the disorder; those who are susceptible to the disorder; and those in whom the disease is to be prevented. A subject is successfully "treated" according to the methods of the present invention if the patient exhibits one or more of the following: a reduction in the number of cancer cells or a complete absence of cancer cells; a reduction in tumor size; inhibition or absence of cancer cell invasion into peripheral organs, including the spread of cancer to soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; a reduction in one or more symptoms associated with the specific cancer; a reduction in morbidity and mortality; and an improvement in quality of life.

[0102] The term "carrier," as used herein, means a pharmaceutically acceptable material, composition, or vehicle, such as, for example, a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, which is involved in or capable of carrying or transporting a subject pharmaceutical compound from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Non-limiting examples of pharmaceutically acceptable carriers, carriers, and / or diluents include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances utilized in pharmaceutical formulations. Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate, magnesium stearate, and polyethylene oxide-polypropylene oxide copolymers, as well as coloring agents, release agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition. 2. Certain Embodiments

[0103] Certain embodiments of the present invention provide double-stranded RNA compositions in which both the antisense and sense strands are composed of linked nucleoside monomers. At least 50 percent of the nucleoside monomers throughout the duplex molecule are ribonucleoside monomers, and some of the ribonucleoside monomers and / or internucleoside linkages contained therein may be modified from those found in natural RNA. The duplex RNA molecules of the present invention further comprise deoxyribonucleoside monomers in one or more deoxyribonucleotide monomer interspersed segments ("ISDs"). One or more ISDs may be found in either the antisense strand or the sense strand, or in both. In some embodiments, each ISD independently consists of one deoxyribonucleotide monomer or at least two, three, four, five, six, seven, eight, nine, or ten consecutive deoxyribonucleotide monomers. In some embodiments, an ISD has at least two consecutive linked deoxyribonucleotide monomers.

[0104] Both the antisense and sense strands of the duplex molecules of the present invention are relatively short, with the antisense strand being the longer of the two, and therefore an "asymmetric short duplex RNA (asdRNA)."

[0105] Exemplary structures and sequences of duplex molecules of the present invention are shown in various figures. For example, in Figure 2A, the ISD is found in the longer antisense strand in all duplex molecules.

[0106] In some embodiments, the length asymmetry between the antisense and sense strands results in at least one overhang in the antisense strand at its 5' end (e.g., the first 3 on the right in Figure 2A) or its 3' end (e.g., the first 10 on the left in Figure 2A) and the other end being a blunt or recessed end. In other embodiments, the antisense strand has overhangs at both ends (e.g., the last 13 on the right in Figure 2A).

[0107] The compositions of the present invention can be used to modulate gene expression or function in eukaryotic cells in at least three ways: (i) contacting a cell with one type of asdRNA molecule or administering it to a subject; (ii) contacting a cell with different types of asdRNA molecules ...

[0108] In certain embodiments, the antisense strand comprises a nucleobase sequence region, referred to as the "targeting region," that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the target segment of the target gene to which it is targeted, including mRNA and non-coding RNA. In certain embodiments, the antisense strand has a nucleobase sequence that comprises the complete complementary sequence of the target segment of the target gene to which it is targeted. In certain embodiments, the antisense strand has a nucleobase sequence that contains at most one, two, or three mismatches when hybridized to the target segment of the target gene to which it is targeted. In certain embodiments, the target gene is selected from mRNA or non-coding RNA involved in mammalian disease. In some embodiments, at least one ISD is located within the targeting region of the antisense strand. In some embodiments, at least one ISD is located more centrally in the antisense strand (i.e., at least 1, 2, 3, 4, or 5 nucleobases away from either end, i.e., starting from position number 2 or more centrally from the end).

[0109] In various embodiments, the antisense strand has a backbone length of 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 linked nucleotide monomers, or its equivalent, or a range sandwiched between any two of the foregoing values, inclusive of the endpoints of the range. For example, some length ranges for the first antisense strand include: 8 to 50 nucleotide monomers; 8 to 36 nucleotide monomers; 8 to 33 nucleotide monomers; 10 to 30 nucleotide monomers; 10 to 29 nucleotide monomers; 12 to 29 nucleotide monomers; 12 to 28 nucleotide monomers; 12 to 26 nucleotide monomers; 12 to 25 nucleotide monomers; 13 to 25 nucleotide monomers; 13 to 24 nucleotide monomers; 13 to 23 nucleotide monomers; 15 to 23 nucleotide monomers; 10 to 36 nucleotide monomers; 12 to 36 nucleotide monomers; 12 to 32 nucleotide monomers; 14 to 36 nucleotide monomers; and at least 8 nucleotide monomers.

[0110] In certain embodiments, the antisense strand is 10 to 36 nucleotide monomers in length (inclusive of both end points of the range). In other words, the antisense strand is 10 to 36 linked nucleobase monomers (inclusive of both end points of the range). In other embodiments, the antisense strand comprises an oligonucleotide consisting of 8 to 100, 10 to 80, 12 to 50, 14 to 30, 15 to 23, 16 to 22, 16 to 21, or 20 linked nucleobases (inclusive of both end points of the range).

[0111] In certain embodiments, the antisense strand consists of 13 to 23 linked nucleoside monomers (inclusive of both endpoints of the range). In certain embodiments, the antisense strand consists of 23 linked nucleoside monomers. In certain embodiments, the antisense strand consists of 20 linked nucleoside monomers. In certain embodiments, the antisense strand consists of 16 linked nucleoside monomers.

[0112] In certain embodiments, the sense strand is substantially complementary to the antisense strand and comprises a nucleobase sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the sequence of the linked region of the antisense oligonucleotide, measured across the entire nucleobase sequence of the sense strand. These substantially complementary sequences from both strands form one or more double-stranded regions. In certain embodiments, the sense strand has a nucleobase sequence that includes the complete complementary sequence of the linked region of the antisense strand. In some embodiments, at least one ISD can be located anywhere in the sense strand. In some embodiments, the ISD is located within the double-stranded region of the sense strand. In some embodiments, the ISD is located more centrally in the sense strand (i.e., at least 1, 2, 3, 4, or 5 nucleobases away from either end, i.e., starting at position number 2 or more centrally from the end). In some embodiments, the ISD need not be located within the sense strand.

[0113] In one aspect, the sense strand has a shorter length than the antisense strand, provided that it is thermodynamically capable of forming a duplex with the antisense strand. In certain embodiments, the sense strand has a length that is about 0.5 to 1 nucleotide shorter than the antisense strand. In certain embodiments, the sense strand has a length that is about 0.25 to about 1 nucleotide shorter than the antisense strand. In certain embodiments, the sense strand is 6 to 35 nucleotide monomers in length (inclusive of both end points of the range). In other words, these sense strands are 6 to 35 linked nucleobases (inclusive of both end points of the range). In other embodiments, the sense strand comprises an oligonucleotide consisting of 13, 4 to 30, 6 to 16, 10 to 20, or 12 to 16 linked nucleobases (inclusive of both end points of the range). In certain such embodiments, the sense strand comprises an oligonucleotide consisting of 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, and 49 linked nucleobases in length, or a range defined by any two of the foregoing values, inclusive of the endpoints of the range. In some embodiments, the sense strand is a sense oligonucleotide.

[0114] In one aspect, the sense strand has a backbone length that is shorter than the antisense strand by the following number of nucleotide monomers: 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, or 38. In various embodiments, the second strand has a backbone length of 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, or 49 linked nucleotide monomers, or an equivalent thereof, or a range between any two of the foregoing values, inclusive of the endpoints of the range. In certain embodiments, for example, some ranges for the sense strand include: 6 to 49 nucleotide monomers; 8 to 46 nucleotide monomers; 8 to 35 nucleotide monomers; 9 to 35 nucleotide monomers; 10 to 46 nucleotide monomers; 10 to 40 nucleotide monomers; 10 to 34 nucleotide monomers; 8 to 32 nucleotide monomers; 8 to 30 nucleotide monomers; 8 to 29 nucleotide monomers; 9 to 29 nucleotide monomers; 9 to 26 nucleotide monomers; 9 to 25 nucleotide monomers; 10 to 29 nucleotide monomers; 10 to 28 nucleotide monomers; 10 to 26 nucleotide monomers; 10 to 25 nucleotide monomers; 11 to 24 nucleotide monomers; 11 to 23 nucleotide monomers; 12 to 23 nucleotide monomers; 13 to 23 nucleotide monomers; a tie of 12 to 22 nucleotide monomers; 13 to 23 nucleotide monomers; a tie of 15 to 23 nucleotide monomers; and at least 6 nucleotide monomers. In certain embodiments, the second strand can have a backbone length of any nucleotide monomer, provided that it is thermodynamically capable of forming a duplex with the first strand.

[0115] In certain embodiments, the sense strand is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide monomers shorter than the antisense strand. In certain embodiments, the sense strand consists of 8 to 23 linked nucleoside monomers (inclusive of both end points of the range). In certain embodiments, the sense strand consists of 13 linked nucleoside monomers. In certain embodiments, the sense strand consists of 14 linked nucleoside monomers.

[0116] In various embodiments of the present invention, the two ends of the antisense strand are in one of the following configurations: a 3'-overhang and a 5'-overhang; a 3'-overhang and a blunt end at the 5'-end; a 5'-overhang and a blunt end at the 3'-end; a 3'-overhang and a 5'-recessed end; or a 5'-overhang and a 3'-recessed end.

[0117] In certain embodiments, the 3'-overhang of the antisense strand has a length 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, or 30 nucleotide monomers. In various embodiments, the 3'-overhang of the antisense strand has a length of 1 to 15, 1 to 10, 1 to 9, 1 to 8, or 1 to 5 nucleotide monomers (inclusive of both end points of the ranges).

[0118] In certain embodiments, the 5'-overhang of the antisense strand has a length 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, or 30 nucleotide monomers. In various embodiments, the 5'-overhang of the antisense strand has a length of 1 to 15, 1 to 10, 1 to 9, 1 to 8, or 1 to 5 nucleotide monomers (inclusive of both end points of the ranges).

[0119] In one embodiment of the invention, the antisense strand has a 3'-overhang of 1 to 15 (inclusive) nucleotide monomers and a 5'-overhang of 1 to 15 (inclusive) nucleotide monomers. In another embodiment, the antisense strand has a 3'-overhang of 1 to 26 (inclusive) nucleotide monomers and a blunt or recessed 5' end. In yet another embodiment, the antisense strand has a 5'-overhang of 1 to 26 (inclusive) nucleotide monomers and a blunt or recessed 3' end.

[0120] In various embodiments of the invention, the two ends of the second (sense) strand are in one of the following configurations: a 3'-overhang and a 5'-recessed end; a 5'-overhang and a 3'-recessed end; a 3'-recessed end and a 5'-recessed end; a 3'-blunt end and a 5'-recessed end; or a 5'-blunt end and a 3'-recessed end. In certain embodiments, the 3'-overhang of the second strand has a length 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, or 25 nucleotide monomers. In various embodiments, the 3'-overhang of the second strand has a length of 1 to 15, 1 to 10, 1 to 9, 1 to 8, or 1 to 5 nucleotide monomers (inclusive of both end points of the ranges). In certain embodiments, the 5'-overhang of the second strand has a length 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, or 25 nucleotide monomers. In various embodiments, the 5'-overhang of the second strand has a length of 1 to 15, 1 to 10, 1 to 9, 1 to 8, or 1 to 5 nucleotide monomers.

[0121] In the asdRNA molecule of the present invention, at least one nucleotide monomer in the first strand and / or second strand can be a modified nucleotide or nucleotide analog, such as a sugar-modified nucleotide, a backbone-modified nucleotide, and / or a base-modified nucleotide. In some embodiments, such backbone-modified nucleotides have at least one internucleoside linkage modified, for example, to include at least one nitrogen or sulfur heteroatom. In some embodiments, the modified internucleoside linkage is or includes a phosphorothioate (P=S) group, a phosphotriester, a methylphosphonate, or a phosphoramidate.

[0122] In certain embodiments, the antisense strand and / or the sense strand comprise at least one modified internucleoside linkage. Such modified internucleoside linkage can be between two ribonucleoside monomers, between two deoxyribonucleoside monomers, or between one deoxyribonucleoside monomer and one ribonucleoside monomer. Alternatively, the phosphate group on at least one of the terminal nucleoside monomers can be modified. In certain embodiments, the internucleoside linkage is a phosphorothioate internucleoside linkage. In certain embodiments, the internucleoside linkage is a thiophosphoramidate internucleoside linkage. In certain embodiments, each internucleoside linkage of the oligonucleotide chain is a phosphorothioate internucleoside linkage. In certain embodiments, all of the internucleoside linkages within a strand (antisense or sense or both) are phosphorothioate internucleoside linkages or a mixture of phosphorothioate and phosphodiester linkages.

[0123] In certain embodiments, the antisense and / or sense strands comprise at least one nucleoside monomer having a modified sugar moiety. Such a nucleoside monomer may be a ribonucleoside or a deoxyribonucleoside monomer.

[0124] In certain embodiments, the 2'-position of the modified sugar moiety is replaced with a group selected from OR, R, halo, SH, SR, NH, NHR, NR, or CN, where each R is independently C-C alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I. In some embodiments, the 2'-position of the modified sugar moiety is replaced with a group selected from allyl, amino, azido, thio, O-allyl, O-C-C 10 Alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), O-CH2-C(=O)-N(R m )(R n ), or O-CH2-C(=O)-N(R1)-(CH2)2-N(R m )(R n ), wherein each R l , R m and R n are independently H or substituted or unsubstituted C1-C 10 In some embodiments, the modified sugar moiety is selected from the group of 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH, 2'-OCHCH, 2'-OCHCHF, and 2'-O(CH)OCH substituents. In some embodiments, the modified sugar moiety is substituted with a bicyclic sugar selected from the group of 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (cEt), and 4'-CH(CHOCH3)-O-2', 4'-C(CH3)(CH3)-O-2', 4'-CH2-N(OCH3)-2', 4'-CH2-ON(CH3)-2', 4'-CH2-N(R)-O-2' (where R is H, C1-C12 alkyl, or a protecting group), 4'-CH2-C(H)(CH3)-2', and 4'-CH2-C-(=CH2)-2'.

[0125] In some embodiments, the modified sugar moiety is selected from the group of 2'-O-methoxyethyl modified sugars (MOE), 4'-(CH2)-O-2' bicyclic sugars (LNA), 2'-deoxy-2'-fluoroarabinose (FANA), and methyl(methyleneoxy) (4'-CH(CH3)-O-2) bicyclic sugars (cEt).

[0126] In some embodiments, the antisense and / or sense strands of the molecules of the invention comprise at least one nucleoside monomer having a modified nucleobase. Such nucleoside monomers may be deoxyribonucleoside or ribonucleoside monomers.

[0127] In some embodiments, modified nucleobases are 5-methylcytosine (5-Me-C), inosine bases, tritylated bases, 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, 1-methyl-pseudo-uracil, 5-halouracil and cytosine; 5-propynyl (—C≡C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases; 6-azo Selected from the group of uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, and 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0128] In certain embodiments, the modified nucleobase in the molecule of the present invention is 5-methylcytosine.In some embodiments, each cytosine base in the molecule of the present invention is 5-methylcytosine.In certain embodiments, the modified nucleobase is 5-methyluracil.In certain embodiments, each uracil is 5-methyluracil.

[0129] In certain embodiments, either the antisense strand or the sense strand, or both strands, of a molecule of the invention comprise linked ribonucleoside monomers. In certain embodiments, the entire strand (antisense or sense) is exclusively composed of linked ribonucleoside monomers. In certain embodiments, the entire sense strand is exclusively composed of linked ribonucleoside monomers. In one feature, either the antisense strand or the sense strand, or both, further comprise an ISD consisting of one or more linked deoxyribonucleoside monomers in addition to linked ribonucleoside monomers. In certain embodiments, one or both strands further comprise an ISD consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 linked deoxyribonucleoside monomers in addition to linked ribonucleoside monomers. In certain aspects, there may be even more ISD segments. The ISDs may be located anywhere on either strand. In some embodiments, one or more ISDs are inserted into segments of ribonucleoside monomers, thereby separating them into multiple segments. In certain embodiments, each ISD independently consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linked deoxyribonucleoside monomers.

[0130] In certain embodiments, at least half of the nucleotides in an asdRNA molecule are ribonucleotide monomers. In various embodiments, at least 50%, 52%, 55%, 58%, 60%, 65%, or 70% of the nucleotides in an asdRNA molecule are ribonucleotide monomers.

[0131] In certain embodiments, the total number of deoxyribonucleotide monomers in an asdRNA molecule is equal to or less than the total number of ribonucleotide monomers in the same asdRNA molecule. In some embodiments, the total number of deoxyribonucleotide monomers in either strand of an asdRNA molecule is equal to or less than the total number of ribonucleotide monomers in the same strand of an asdRNA molecule. In certain embodiments, the total number of deoxyribonucleotide monomers in the first strand of an asdRNA molecule is equal to or less than the total number of ribonucleotide monomers in the same first strand of an asdRNA molecule. The total number of deoxyribonucleotide monomers in the second strand of an asdRNA molecule is equal to or less than the total number of ribonucleotide monomers in the same second strand of an asdRNA molecule. In certain embodiments, the total number of deoxyribonucleotide monomers in an asdRNA molecule is equal to or less than the total number of ribonucleotide monomers in the same asdRNA molecule, but the total number of deoxyribonucleotide monomers in one strand of an asdRNA molecule may be greater than the total number of ribonucleotide monomers in the same strand of an asdRNA molecule.

[0132] In certain embodiments, at least one or each of the linked deoxyribonucleoside monomers of an ISD is a modified deoxyribonucleotide or deoxyribonucleotide analog. The deoxyribonucleotide may be modified in the same or similar manner as follows: to have a modified internucleoside linkage, a modified sugar moiety, and / or a modified nucleobase.

[0133] In some embodiments, the sugar moiety of the deoxyribonucleotide monomer is either a sugar moiety of a naturally occurring deoxyribonucleotide (2-H) or 2'-deoxy-2'-fluoroarabinose (FANA).

[0134] In some embodiments, the sugar moiety of the ribonucleotide monomer is selected from the group consisting of naturally occurring ribonucleotides (2-OH), 2'-F modified sugars, 2'-OMe modified sugars, 2'-O-methoxyethyl modified sugars (MOE), 4'-(CH2)-O-2' bicyclic sugars (LNA), and methyl(methyleneoxy) (4'-CH(CH3)-O-2) bicyclic sugars (cEt).

[0135] In certain embodiments, at least one deoxyribonucleoside monomer of each ISD in the antisense strand, sense strand, or both strands has a modified sugar moiety of 2'-deoxy-2'-fluoroarabinose (FANA). In another embodiment, all of the nucleosides in the ISD have a modified sugar moiety such as FANA. In a further embodiment, all of the nucleosides in the ISD are naturally occurring deoxyribonucleosides. In one embodiment, all of the nucleosides in the ISD are naturally occurring deoxyribonucleosides or have a modified sugar moiety such as FANA. In certain embodiments, at least one or each ribonucleoside monomer in the antisense strand, the sense strand, or both strands has a modified sugar moiety selected from the group consisting of 2'-O-methoxyethyl modified sugar (MOE), 4'-(CH2)-O-2' bicyclic sugar (LNA), and methyl(methyleneoxy) (4'-CH(CH3)-O-2) bicyclic sugar (cEt).

[0136] In certain embodiments, each internucleoside linkage within the deoxyribonucleoside monomer of each ISD is a phosphorothioate linkage. In certain embodiments, each internucleoside linkage within the deoxyribonucleoside monomer of each ISD is a natural phosphate linkage without a phosphorothioate modification.

[0137] In certain embodiments, each deoxyribonucleoside monomer of each ISD has a FANA where each cytosine is a 5-methylcytosine. In certain embodiments, each deoxyribonucleoside monomer of each ISD has a FANA where each cytosine is a 5-methylcytosine and each internucleoside linkage is a phosphorothioate linkage.

[0138] In certain embodiments, the molecules of the invention have either an antisense strand or a sense strand composed of ribonucleoside monomers in which each internucleoside linkage is a phosphorothioate linkage. In certain embodiments, the molecules of the invention have either an antisense strand or a sense strand composed of ribonucleoside monomers in which each internucleoside linkage is a natural phosphate linkage without a phosphorothioate modification.

[0139] In certain embodiments, a molecule of the invention comprises a sense strand, wherein each nucleotide monomer of the sense strand contains the same modification as the complementary nucleotide monomer of the antisense strand.

[0140] Exemplary structures of molecules of the invention having antisense and sense oligonucleotide strands are shown in FIGS. 2A, 3A, 4A, and 5.

[0141] In certain embodiments, asymmetric short duplex RNA and at least one ISD in the antisense strand of the duplex molecule enable potent gene silencing. The data presented in all of the examples below suggest that a new platform technology based on asymmetric duplex RNA with at least one ISD in the antisense oligoribonucleotide enables extremely potent gene silencing. Further research was conducted on the SAR (structure-activity relationship) characteristics of asdRNA, including length motifs, ISD motifs, and various modifications. This research helps define various structural design elements that can affect gene silencing activity. Such SAR factors are important for designing optimized gene silencers that target the diverse sequences and structures of more than 100,000 different mRNAs and even more non-coding RNAs in typical mammalian cells. Our data on the gene silencing activity and SAR factors of asdRNA suggest that the gene silencing characteristics of asdRNA are significantly different from those of siRNA and ASO, indicating a novel and unique gene silencing mechanism that has yet to be elucidated.

[0142] In certain embodiments, the molecules of the present invention can be stabilized against degradation by at least one chemical modification or secondary structure.Sense oligonucleotide strand and antisense oligonucleotide strand can have unmatched nucleotide monomers or imperfectly matched nucleotide monomers.Sense oligonucleotide strand and / or antisense oligonucleotide strand can have one or more nicks (cuts in the nucleic acid backbone), gaps (fragmented strands with one or more missing nucleotides), and modified nucleotides or nucleotide analogs.Not only can any or all of the nucleotide monomers in the sense and antisense oligonucleotide strands be chemically modified, but each strand can also be conjugated with one or more moieties or ligands, for example, with a moiety or ligand selected from peptides, antibodies, antibody fragments, polymers, polysaccharides, lipids, hydrophobic moieties or molecules, cationic moieties or molecules, lipophilic compounds or oligonucleotide moieties, cholesterol, GalNAc, and aptamers, to enhance its functionality.

[0143] In certain embodiments, the double-stranded region of the duplex molecule of the present invention does not contain any mismatches or bulges, and the two strands are fully complementary to each other in the double-stranded region. In another embodiment, the double-stranded region of the duplex contains mismatches and / or bulges.

[0144] In certain embodiments, the target is mRNA, pre-mRNA, mt-mRNA, or non-coding RNA involved in mammalian disease. In certain embodiments, the target is mRNA. In certain embodiments, the target is pre-mRNA. In certain embodiments, the target is non-coding RNA, such as microRNA and lncRNA. In certain embodiments, the target is mt-mRNA. If the antisense strand is substantially complementary to the target sequence, it can hybridize to occupy the target and inactivate the target gene.

[0145] 3. Unmatched or mismatched regions The complementary region between the antisense strand and the sense strand of the asdRNA of the present invention can have at least one unmatched region or an incompletely matched region, for example, containing one or more mismatches.Mismatches in the sense strand can be desirable to reduce off-target effects, or can enable other features for the asdRNA.

[0146] As known to those skilled in the art, it is possible to introduce mismatched bases without losing activity.Similarly, the antisense strand of asdRNA of the present invention can comprise unmatched or mismatched region when base-pairing with target RNA.The mismatch in antisense strand may be desirable to reduce off-target effect, or may enable other features for asdRNA.

[0147] 4. Modifications Nucleoside monomers are base-sugar compositions. The nucleobase (also known as base) portion of a nucleoside monomer is usually a heterocyclic base moiety. Nucleotide monomers are nucleoside monomers that further contain a phosphate group covalently linked to the sugar portion of the nucleoside. For nucleotide monomers containing a pentofuranosyl sugar, the phosphate group may be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. Oligonucleotides are formed by covalently linking adjacent nucleoside monomers to each other to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.

[0148] Modifications of the asdRNA molecules, antisense strands, and / or sense strands of the present invention include substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified asdRNA, antisense strands, and / or sense strands are sometimes preferred over native forms due to desirable properties, such as increased inhibitory activity, enhanced cellular uptake, enhanced strand affinity, solubility, reduced nonspecific interactions, and resistance to RNase degradation or other enhanced stability. Therefore, results equivalent to those of short antisense strands with such chemically modified nucleoside monomers can often be achieved. One or more of the natural nucleotides in the antisense and sense strands of the present invention can be replaced with modified nucleotides or nucleotide analogs. Substitutions can occur anywhere in the antisense and sense strands.

[0149] Modifications of oligonucleotide molecules have been investigated to improve the stability of various oligonucleotide molecules, including antisense oligonucleotides, ribozymes, aptamers, and RNAi (Chiu and Rana, 2003; Czauderna et al., 2003; de Fougerolles et al., 2007; Kim and Rossi, 2007; Mack, 2007; Zhang et al., 2006; Schrnidt, 2007; Setten RL et al., 2020; Crooke ST et al., 2018; and Roberts TC et al., 2020).

[0150] Any stabilizing modification known to those skilled in the art can be used to improve the stability of oligonucleotide molecules.In oligonucleotide molecules, chemical modifications can be introduced into the phosphate backbone (e.g., phosphorothioate linkage), sugar (e.g., locked nucleic acid, glycerol nucleic acid, cEt, 2'-MOE, 2'-fluorouridine, 2'-O-methyl), and / or base (e.g., 2'-fluoropyrimidine).

[0151] Some examples of such chemical modifications are summarized in the following sections.

[0152] In various embodiments, the modified nucleotide or nucleotide analog is a sugar-modified nucleotide, a backbone-modified nucleotide, and / or a base-modified nucleotide.

[0153] 4.1 Modified Internucleoside Linkages or Backbone-Modified Nucleotides The naturally occurring internucleoside linkage in RNA and DNA is a 3' to 5' phosphodiester linkage. The asdRNA molecules of the present invention having one or more modified, i.e., non-naturally occurring, internucleoside linkages in one or both strands may be chosen over corresponding molecules having only naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, increased affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0154] The oligonucleotide chain having modified internucleoside linkages includes not only internucleoside linkages that retain phosphorus atoms, but also internucleoside linkages that do not have phosphorus atoms.In some embodiments, phosphodiester internucleoside linkages are modified to include at least nitrogen and / or sulfur heteroatoms.Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, thiophosphoramidate and phosphorothioate.Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known.

[0155] In one embodiment, the modified nucleotide or nucleotide analog is a backbone-modified nucleotide. The backbone-modified nucleotide may have a modified phosphodiester internucleoside linkage. In a further embodiment, the backbone-modified nucleotide is a phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage.

[0156] 4.2 Modified sugar moieties The antisense and / or sense strands of the invention may optionally contain one or more nucleoside monomers in which the sugar group has been modified. Such sugar-modified nucleoside monomers may confer enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the strand. In certain embodiments, the nucleoside monomer comprises a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and 2' substituents), bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNAs), and the modification of ribosyl ring oxygen atoms to S, N(R), or C(R1)(R2) (where R, R1, and R2 are each independently H, C1-C2). 12 Examples of chemically modified sugars include 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO2008 / 101157, published August 21, 2008, for other disclosed 5',2'-disubstituted nucleosides), or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2' position (see U.S. Patent Application Publication No. 2005-0130923, published June 16, 2005), or alternatively, 5' substitution of BNAs (see PCT International Application WO2007 / 134181, published November 22, 2007, in which LNAs are substituted, for example, with a 5'-methyl or 5'-vinyl group).

[0157] Examples of nucleoside monomers having modified sugar moieties include, but are not limited to, nucleosides containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH, 2'-OCHCH, 2'-OCHCHF, and 2'-O(CH)OCH substituents. The substituent at the 2' position can be any of allyl, amino, azido, thio, O-allyl, O-C1-C2 10and O—CH—C(═O)—N(R)(R), and O—CH—C(═O)—N(R)—(CH)—N(R)(R), where each R, R, and R is independently H or a substituted or unsubstituted C to C 10 It is alkyl.

[0158] Bicyclic nucleosides are modified nucleosides having a bicyclic sugar moiety. Examples of bicyclic nucleic acids (BNAs) include, but are not limited to, nucleosides comprising a bridge between the 4'-ribosyl ring atom and the 2'-ribosyl ring atom. In certain embodiments, the asdRNA, antisense strand, and / or sense strand provided herein comprise one or more BNA nucleosides in which the bridge comprises one of the following formulas: 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' and 4'-CH(CHOCH3)-O-2' (and analogs thereof, see U.S. Patent No. 7,399,845 issued July 15, 2008); 4'-C(CH3)(CH3)-O-2' (and analogs thereof). 4'-CH-N(OCH)-2' (and its analogs, see PCT / US2008 / 064591, published December 11, 2008, as WO / 2008 / 150729); 4'-CH-ON(CH)-2' (see U.S. Patent Application Publication No. 2004-0171570, published September 2, 2004); 4'-CH-N(R)-O-2' (wherein R is H, C1-C 12alkyl, or a protecting group) (see U.S. Pat. No. 7,427,672, issued Sep. 23, 2008); 4'-CH2-C(H)(CH3)-2' (see Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C-(=CH2)-2' (and analogs thereof, see PCT / US2008 / 066154, published Dec. 8, 2008, published as WO2008 / 154401).

[0159] In certain embodiments, the bicyclic nucleoside is: (A) α-L-methyleneoxy(4′-CH2-O-2)BNA (B) β-D-methyleneoxy(4′-CH2-O-2)BNA These include, but are not limited to, (C) ethyleneoxy (4'-(CH)-O-2') BNAs, (D) aminooxy (4'-CH-ON(R)-2') BNAs, (E) oxyamino (4'-CH-N(R)-O-2) BNAs, (F) methyl (methyleneoxy) (4'-CH(CH)-O-2) BNAs (referred to as constrained ethyl or cEt), (G) methylene-thio (4'-CH-S-2') BNAs, (H) methylene-amino (4'-CH-N(R)-2') BNAs, (I) methyl carbocyclic (4'-CH-CH(CH)-2) BNAs, (J) propylene carbocyclic (4'-(CH)-2') BNAs, and (K) vinyl BNAs.

[0160] In certain embodiments, the modified nucleotide or nucleotide analog is a sugar-modified ribonucleotide in which the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH, NHR, NR, and CN, where each R is independently selected from the group consisting of C-C alkyl, alkenyl, and alkynyl, and halo is selected from the group of F, Cl, Br, and I. In certain embodiments, the sugar-modified ribonucleotide is selected from the group of 2'-OMe-modified nucleotides, 2'-F-modified nucleotides, 2'-O-methoxyethyl (2'MOE)-modified nucleotides, LNA (locked nucleic acid)-modified nucleotides, GNA (glycerol nucleic acid)-modified nucleotides, and cEt (constrained ethyl)-modified nucleotides. In certain embodiments, the sugar-modified deoxyribonucleotide is a FANA-modified deoxyribonucleotide.

[0161] The molecule of the present invention can be stabilized by adopting chemical modification at the 2' position of ribose, such as 2'-O-methylpurine and 2'-fluoropyrimidine, which increases the resistance to endonuclease activity in serum.The position for introducing modification should be carefully selected so as to avoid the significant reduction of the silencing / regulating efficacy of molecule.In certain embodiments, the first nucleotide monomer adjacent to the 5'-end nucleotide monomer of antisense strand is 2'-fluoro-ribonucleotide.

[0162] 4.3 Modified Nucleobases The antisense and / or sense strands in asdRNA molecules may also have nucleobase (or base) modifications or substitutions. The nucleobase (or base) modifications or substitutions are structurally distinct from naturally occurring or synthetic unmodified nucleobases, but functionally interchangeable. Both natural and modified nucleobases can participate in hydrogen bonds. Such nucleobase modifications may confer nuclease stability, binding affinity, or some other beneficial biological properties to asdRNA molecules. Modified nucleobases include synthetic and natural nucleobases, such as 5-methylcytosine (5-Me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of antisense and sense strands. For example, 5-methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

[0163] Additional modified nucleobases include 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, 1-methylpseudouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine; 5-propynyl (-C≡C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases; 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0164] Heterocyclic base moiety can comprise purine or pyrimidine base replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.The nucleic acid base that is particularly useful for increasing the binding affinity of antisense and sense strands includes 5-substituted pyrimidine, 6-azapyrimidine and N-2, N-6 and O-6 substituted purine, and said substituted purine includes 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

[0165] In certain embodiments, the modified nucleotide or nucleotide analog is a base-modified nucleotide. In certain embodiments, the modified nucleotide or nucleotide analog has an unusual or modified base. In certain embodiments, the modified base is 5-methylcytosine (5'-Me-C). In certain embodiments, each cytosine is a 5-methylcytosine. In certain embodiments, the modified base is 5-methyluracil (5'-Me-U). In certain embodiments, each uracil is a 5-methyluracil.

[0166] Any modified nucleotide or analog that may aid in stability or affinity can be made without departing from the spirit and scope of the present invention. Some examples of such chemical modifications are the same as those summarized above.

[0167] 5. Pharmaceutical Compositions In some embodiments, the present invention also provides pharmaceutical formulations comprising the asdRNA of the present invention or a pharmaceutically acceptable derivative thereof and at least one pharmaceutically acceptable excipient or carrier. As used herein, "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in "Remington: The Science and Practice of Pharmacy, Twentieth Edition," Lippincott Williams & Wilkins, Philadelphia, PA, incorporated herein by reference. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the asdRNA molecule, use thereof in the compositions is contemplated.

[0168] Examples of pharmaceutically acceptable carriers that can be used with the molecules of the invention include, but are not limited to, pharmaceutical carriers, positively charged carriers, liposomes, lipid nanoparticles, protein carriers, hydrophobic moieties or molecules, cationic moieties or molecules, GalNAc, polysaccharides, polymers, nanoparticles, nanoemulsions, cholesterol, lipids, lipophilic compounds or moieties, and lipids.

[0169] In certain embodiments, the present invention provides a method of treatment comprising administering a therapeutically effective amount of a pharmaceutical composition to a subject in need thereof. In certain embodiments, the pharmaceutical composition is administered by a route selected from the group consisting of intravenous injection (iv), subcutaneous injection (sc), orally (po), intramuscular (im) injection, oral administration, inhalation, topical, intrathecal, and other localized administration. In other embodiments, the therapeutically effective amount is 1 ng to 1 g per day, 100 ng to 1 g per day, or 1 μg to 1000 mg per day.

[0170] Methods for formulation are described in PCT International Application PCT / US02 / 24262 (WO03 / 01 1224), U.S. Patent Application Publication Nos. 2003 / 0091639 and 2004 / 0071775, each of which is incorporated herein by reference.

[0171] The asdRNA molecules of the present invention are administered in a suitable dosage form prepared by combining a therapeutically effective amount (e.g., an effective level sufficient to achieve the desired therapeutic effect by inhibiting tumor growth, killing tumor cells, treating or preventing a cell proliferative disorder, etc.) of the asdRNA molecules of the present invention (as the active ingredient) with standard pharmaceutical carriers or diluents according to conventional procedures (i.e., by producing a pharmaceutical composition of the present invention).

[0172] These procedures may include mixing, granulating, and compressing or dissolving the ingredients, as appropriate, to achieve the desired preparation. In another embodiment, a therapeutically effective amount of an asdRNA molecule is administered in a suitable dosage form without standard pharmaceutical carriers or diluents. In some embodiments, a therapeutically effective amount of a duplex molecule of the present invention is administered in a suitable dosage form. Pharmaceutically acceptable carriers include solid carriers such as lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, and the like. Exemplary liquid carriers include syrup, peanut oil, olive oil, water, and the like. Similarly, carriers or diluents include time-delay materials known in the art, such as glyceryl monostearate or glyceryl distearate, alone or with wax, ethylcellulose, hydroxypropylmethylcellulose, methyl methacrylate, and the like. Other fillers, excipients, flavorants, and other additives, such as those known in the art, may also be included in pharmaceutical compositions according to the present invention.

[0173] The pharmaceutical composition of the present invention can be prepared in a generally known manner, for example, by conventional mixing, dissolving, granulating, making sugar-coated tablets, elutriating, emulsifying, encapsulating, entrapping or lyophilizing process.Pharmaceutical composition can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and / or auxiliary agents that facilitate the processing of sense oligonucleotide and antisense oligonucleotide into pharmaceutical preparations that can be used.Of course, suitable formulation depends on the selected route of administration.

[0174] The compositions, compounds, combinations or pharmaceutical compositions of the present invention can be administered to subjects by many of the well-known methods currently used in chemotherapy treatment.For example, for the treatment of cancer, the asdRNA molecules of the present invention can be directly injected into tumors, injected into the bloodstream or body cavities, or taken orally, or applied through the skin using a patch.For the treatment of psoriasis, systemic administration (e.g., oral administration) or topical administration to the affected area of ​​the skin is the preferred administration route.The selected dose should be sufficient to constitute effective treatment, but not so high as to cause unacceptable side effects.The patient's condition (e.g., cancer, psoriasis, etc.) and health status should be closely monitored during and for a reasonable period after treatment.

[0175] 6.Usefulness 6.1 How to use The present invention also provides a method for modulating gene expression or function in a cell or organism. The cell can be a eukaryotic cell, such as a mammalian cell. The method includes contacting the cell or organism with an asdRNA molecule disclosed herein under conditions that allow selective gene silencing, and mediating selective gene silencing exerted by the asdRNA molecule on a target nucleic acid having a sequence portion substantially complementary to the antisense strand. The target nucleic acid can be RNA, such as mRNA, pre-mRNA, mt-mRNA, or non-coding RNA, and such RNA encodes a protein involved in a disease or controls part of a biological pathway involved in a disease.

[0176] In some embodiments, the contacting step comprises introducing the asdRNA molecule into target cells in culture or into target cells in an organism capable of selective gene silencing. In further embodiments, the introducing step comprises mixing, transfection, lipofection, infection, electroporation, or other delivery techniques. In other embodiments, the introducing step comprises administering by iv, sc, intrathecal, p.o., inhalation, topical, or other clinically acceptable administration method using a pharmaceutically acceptable excipient, carrier, or diluent selected from the group consisting of pharmaceutical carriers, positively charged carriers, liposomes, lipid nanoparticles, protein carriers, polymers, nanoparticles, nanoemulsions, lipids, N-acetyl-galactosamine (GalNAc), lipophilic compounds or moieties, and lipoids.

[0177] In certain embodiments, the silencing method is used to determine the function or utility of a gene in a cell or organism.

[0178] In some embodiments, the gene or RNA targeted by the compositions of the present invention is associated with or involved in a disease, such as a human disease or animal disease, a pathological condition, or an undesirable condition. In a further embodiment, the target gene or RNA is of a pathogenic microorganism. In yet a further embodiment, the target gene or RNA is of viral origin. In another embodiment, the target gene or RNA is tumor-associated.

[0179] In alternative embodiments, the gene or RNA targeted by the compositions of the invention is a gene or RNA associated with, or more specifically involved in, a cancer, an autoimmune disease, an inflammatory disease, a degenerative disease, an infectious disease, a proliferative disease, a metabolic disease, an immune-mediated disorder, an allergic disease, a dermatological disease, a malignant disease, a gastrointestinal disorder, a hepatic disorder, a respiratory disorder, a cardiovascular disorder, a dermatological disorder, a nephrological disorder, a rheumatoid disorder, a neurological disorder, a psychiatric disorder, an endocrine disorder, or an aging-related disorder or disease.

[0180] 6.2 Treatment method The present invention also provides methods for treating or preventing various diseases or conditions, including those summarized for ASOs and siRNAs (Czech, 2006; de Fougerolles et al., 2007; Dykxhoorn et al., 2003; Kim and Rossi, 2007; Mack, 2007; Crooke ST et al., 2018; Setten RL et al., 2019; Roberts TC et al., 2020). The method includes administering an effective amount of an asdRNA molecule to a subject in need thereof under conditions that allow for the desired gene inhibition described in the section immediately above.

[0181] In an exemplary embodiment, a pharmaceutical composition having an asdRNA molecule and a pharmaceutically acceptable excipient, carrier, or diluent is administered in a therapeutically effective amount to a patient in need thereof to treat or prevent a disease or undesirable condition.

[0182] In some embodiments, the present invention can be used as cancer treatment or to prevent cancer.AsdRNA compositions can be used to silence or knock down genes involved in cell proliferation disorders or malignant diseases.Examples of these genes are k-Ras, β-catenin, and Stat3.These oncogenes are active and related to many human cancers.

[0183] The novel compositions of the present invention can also be used to treat or prevent ophthalmic diseases (e.g., age-related macular degeneration (AMD) and diabetic retinopathy (DR)); infectious diseases (e.g., HIV / AIDS, hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV), herpes simplex virus (HSV), RCV, cytomegalovirus (CMV), dengue fever, West Nile virus); respiratory diseases (e.g., respiratory syncytial virus (RSC), asthma, cystic fibrosis); neurological diseases (e.g., Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), spinal cord injury, Parkinson's disease, Alzheimer's disease, pain); cardiovascular diseases; metabolic disorders (e.g., hyperlipidemia, hypercholesterolemia, and diabetes); genetic disorders; and inflammatory conditions (e.g., inflammatory bowel disease (IBD), arthritis, rheumatoid diseases, autoimmune disorders), dermatological diseases.

[0184] In alternative embodiments, the method of administration is a route selected from the group of intravenous injection (iv), subcutaneous injection (sc), orally (po), intrathecal, inhalation, topical, and other localized administration. [Example]

[0185] Examples are provided below to further illustrate different features of the present invention. The examples also illustrate useful methodologies for practicing the invention. These examples do not limit the claimed invention. Methods and Materials cell culture

[0186] DLD1 cells were purchased from ATCC. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% inactivated fetal bovine serum (FBS). HepaRG cells were grown in Williams' medium supplemented with 10% FBS, 10 mg / ml hydrocortisone, and 4 mg / ml human recombinant insulin. Other suitable commercially available cell lines can be purchased and used, as known to those skilled in the art.

[0187] Transfection of asdRNA into cells 24 hours before transfection, DLD1 cells, HepaRG cells, or other commonly used cell lines were seeded in 6-well plates (1 x 105 cells / 2 mL / well). AsdRNA was transfected at different final concentrations (e.g., 100 pM, 200 pM, 1 nM, 3 nM, 5 nM, 10 nM, or 30 nM) using Lipofectamine® RNAiMAX (Thermo Fisher Scientific, USA) as described in the manufacturer's instructions. Briefly, asdRNA and RNAiMAX were incubated in serum-free OPTI-MEM (Thermo Fisher Scientific) for 20 minutes and then added to the cells along with the culture medium.

[0188] quantitative PCR Cells transfected with the indicated asdRNAs were harvested 48 hours posttransfection. RNA was isolated with TRIZOL and subjected to qRT-PCR using TaqMan one-step RT-PCR reagents and the CTNNB1 assay (Thermo Fisher Scientific) for β-catenin mRNA, the APOCIII assay for APOCIII mRNA, and the APOB assay for APOB mRNA. GAPDH mRNA levels were used as an internal control.

[0189] Target sequence

[0190] To investigate the gene silencing effect of the asdRNA disclosed in the present invention, asdRNAs were designed and prepared to target different genes. Exemplary target genes and target sequences designed and used are shown in Figure 1, and exemplary sequences of the corresponding antisense strands of the asdRNAs are also shown in Figure 1.

[0191] Example 1 Structure-activity relationship (SAR) studies on asdRNAs with ISDs located exclusively at the AS and pure RNA SSs of various lengths and positions Figure 2A shows various structures of a series of asdRNA embodiments in which the ISD is found exclusively in the AS. The ISD-containing antisense strand (AS) is kept constant while the annealing position and length of the sense strand (SS), which is composed exclusively of linked ribonucleoside monomers, are varied (labeled sdRNA-a1 to -a33 in Figure 2A). sdRNA-a1 to -a33 were designed to target the APOCIII gene (sequences shown in Figure 2B). Single-stranded antisense oligonucleotides (ASOs) with the same structure and sequence of the antisense strand of the asdRNA were also designed as the corresponding single-stranded ASOs for comparison. The gene silencing activity of these asdRNAs and the corresponding single-stranded ASOs was tested at 100 pM in HepaRG cells.

[0192] In Figure 2A, all letters "D" in the structures shown represent DNA residues or deoxyribonucleotide monomers; all letters "R" in the structures shown represent RNA residues or ribonucleotide monomers, including 2'-MOE-modified RNA residues or 2'-MOE-modified ribonucleotide monomers; all letters "rR" in the structures shown represent RNA residues or ribonucleotide monomers, including naturally occurring RNA residues or ribonucleotide monomers; and all "*" in the structures shown represent PS (phosphorothioate internucleoside linkages).

[0193] In Figure 2B, all lowercase letters "a, c, g, t" in the sequence represent DNA residues, all uppercase letters "A, C, G, U" in the sequence represent 2'-MOE modified RNA residues, and all underlined uppercase letters in the sequence [ka] represents an RNA residue, where all "U"s are 5-methyluridine 2'-MOE RNA residues, and all [ka] are 5-methyluridine RNA residues, all "C"s are 5-Me-cytosine 2'-MOE RNA residues, all "c"s are 5-Me-cytosine DNA residues, and all [ka] is a 5-Me-cytosine RNA residue; all "*" in the sequence represent PS (phosphorothioate internucleoside linkage).

[0194] The gene silencing results are shown in Figure 2C. This figure suggests that all the designed asdRNAs have high potency of gene silencing activity against APOCIII at very low concentrations (picomolar level), and are much more potent and effective than the corresponding single-stranded ASO, ISIS304801, which is optimized with the most advanced state-of-the-art know-how.

[0195] Example 2 Structure-activity relationship (SAR) studies on asdRNAs with ISDs in both SS and AS Figure 3A shows the different structural designs of another series of asdRNA embodiments. In these asdRNAs, the SS containing the ISD was kept constant, but the number of deoxyribonucleotide monomers of the ISD located at various positions within the antisense strand was varied (labeled sdRNAs b1-b4) (structures and sequences shown in Figure 3A). Single-stranded antisense oligonucleotides with identical structures and sequences to the antisense strands of asdRNAs b1-b4 were also designed as corresponding ASOs for each asdRNA for comparison. The gene silencing activity of asdRNAs b1-b4 and their corresponding ASOs designed to target APOCIII was tested in HepaRG cells at 100 pM (comparison results are shown in Figure 3B).

[0196] In Figure 3A, all letters "D", "R" in the structures shown represent the same as in Figure 2A, and all lowercase letters "a, c, g, t", uppercase letters "A, C, G, U" and "*" in the sequences shown represent the same as in Figure 2B.

[0197] The results suggest that all designed asdRNAs with at least one ISD within the AS have extremely potent gene silencing activity at very low concentrations (picomolar level), and are significantly more potent and effective than the corresponding ASOs.

[0198] Example 3 SAR studies on asdRNAs bearing ISD in AS with unmodified internucleoside linkages

[0199] Figure 4A shows exemplary structural designs for a series of embodiments of asdRNAs targeting APOB genes, each with an ISD located exclusively at the AS. Each internucleoside linkage between adjacent nucleoside monomers in these asdRNA molecules is a naturally occurring internucleoside linkage. By keeping the AS constant and varying the annealing position and length of the SS, which is composed exclusively of ribonucleoside monomers (labeled sdRNA-c1 to -c8), the gene silencing effects of such structural variations were tested at 5 nM in HepaRG cells. The results are shown in Figure 4B.

[0200] In Figure 4A, all letters "D," "R," and "rR" in the structures shown represent the same as in Figure 2A, and all lowercase letters "a, c, g, t," uppercase letters "A, C, G, U," and underlined uppercase letters in the sequences shown of the asdRNAs. [ka] represents the same as in Figure 2B.

[0201] Specifically, at 5 nM per sample dose, sdRNA-c1–c8 (structure and sequence shown in Figure 4A) exhibited strong gene silencing activity against the intended target, the APOB gene, in HepaRG cells.

[0202] Example 4 Gene silencing efficacy of asdRNA targeting β-catenin

[0203] The structures and sequences of the designed and used asdRNAs targeting β-catenin are listed in Figure 5. The gene silencing efficacy of asdRNAs targeting β-catenin was tested in DLD1 cells at 100 pM, 200 pM, 1 nM, 3 nM, 10 nM, and 30 nM. The results are shown in Figure 5. In Figure 5, all lowercase letters "a, c, g, t" in the sequences represent DNA residues, all uppercase letters "A, C, G, U" in the sequences represent RNA residues, including 2'-MOE modified RNA residues, letters "rG, rA, rC, rU" in the sequences represent RNA residues, and all "*" in the sequences represent PS (phosphorothioate internucleoside linkages).

[0204] The results in Examples 1 to 4 strongly suggest that asdRNAs designed according to the principles of the present invention can achieve high gene silencing efficacy against different genes targeted by different targeting sequence motifs.

[0205] Another example of testing the gene silencing effect of the asdRNA disclosed in the present invention, in which the asdRNA is designed to target pre-mRNA in the nucleus, lncRNA in the nucleus, and mt-mRNA in the mitochondria, was carried out under the same method as in the above example, and the quantitative PCR results all showed that the asdRNA designed according to the principle of the present invention can achieve high gene silencing efficacy, while the corresponding aiRNA / siRNA designed to target the same RNA in the nucleus and mitochondria cannot exhibit gene silencing activity.

[0206] equivalent The representative examples are intended to serve to illustrate the invention and are not intended to, and should not be construed as, limiting the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the complete contents of this document, including the examples and references to scientific and patent literature contained herein. The examples contain important additional information, exemplification and guidance that may be adapted to the practice of this invention in its various embodiments and equivalents thereof.

[0207] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are now described. The methods described herein can be carried out in any order that is logically possible, in addition to the specific order disclosed.

[0208] Incorporation by Reference References to and citations of other documents, such as patents, patent applications, patent publications, periodicals, books, academic papers, and web content, have been made in this disclosure. All such documents are hereby incorporated by reference in their entirety for all purposes. Any material or portion thereof that is stated to be incorporated by reference herein but that contradicts existing definitions, descriptions, or other disclosed material expressly set forth herein, is incorporated only to the extent that no contradiction arises between the incorporated material and the disclosed material. In the event of a conflict, the conflict should be resolved in favor of the present disclosure as the preferred disclosure. References [ka] [ka]

Claims

1. 1. An asymmetric short double-stranded RNA (asdRNA) molecule comprising a first strand and a second strand, each strand comprising linked nucleotide monomers, the second strand is shorter than the first strand; the first strand is substantially complementary to a segment targeted by at least one targeting region of an RNA; the second strand is substantially complementary to the first strand and forms at least one double-stranded region with the first strand; the asdRNA molecule comprises at least one deoxyribonucleotide monomer interspersed segment (ISD) in the first strand or the second strand or both strands, the interspersed segment comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deoxyribonucleotide monomers; the total number of deoxyribonucleotide monomers is less than or equal to the total number of ribonucleotide monomers in the asdRNA molecule; asdRNA molecule.

2. The asdRNA molecule of claim 1, wherein the asdRNA having the ISD has at least one improved gene modulation or pharmaceutical property compared to a corresponding asymmetric RNA duplex without the ISD.

3. wherein said at least one improved gene modulation or pharmaceutical property is: (a) inducing gene silencing at picomolar concentrations, e.g., 500 pM, 300 pM, 200 pM, 100 pM or even lower; (b) enabling gene silencing in the cytoplasm as well as in the nucleus and mitochondria; (c) eliminating or reducing interference with endogenous microRNA function; or (d) Tolerating a wider range of chemical modifications, including non-RNA-like nucleotide modifications or substitutions. (e) Lower synthesis costs and improved stability The asdRNA molecule of claim 2, comprising:

4. 2. The asdRNA molecule of claim 1, wherein the at least one ISD comprises at least four consecutive deoxyribonucleotide monomers.

5. 2. The asdRNA molecule of claim 1, wherein the at least one ISD is located within at least one targeted region of the first strand.

6. 6. The asdRNA molecule of claim 5, wherein the at least one ISD comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive deoxyribonucleotide monomers.

7. 2. The asdRNA molecule of claim 1, wherein the at least one ISD is located within at least one double-stranded region of the second strand.

8. 8. The asdRNA molecule of claim 7, wherein the at least one ISD comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive deoxyribonucleotide monomers.

9. 9. The asdRNA molecule of claim 1, wherein the at least one ISD is located within at least one targeted region of the first strand and within at least one double-stranded region of the second strand.

10. 10. The asdRNA molecule of any one of claims 1 to 9, wherein the first strand is at least 70%, 80%, 85%, 90%, 95% or fully complementary to the targeted segment of the targeted RNA.

11. 11. The asdRNA molecule of claim 10, wherein the first strand has a length selected from the group consisting of 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50 nucleotide monomers.

12. The first chain is a) 8 to 50 nucleotide monomers; b) 10 to 36 nucleotide monomers; c) 12 to 36 nucleotide monomers, and d) 12 to 25 nucleotide monomers 11. The asdRNA molecule of claim 9 or 10, having a length selected from the group consisting of:

13. 13. The asdRNA molecule of any one of claims 1 to 12, wherein the second strand comprises a substantially complementary region that is at least 70%, 75%, 80%, 85%, 90%, 95% complementary or fully complementary to at least one region of the first strand.

14. (a) the second strand is shorter than the first strand by a number of monomers selected 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, and 38; (b) the second strand has a length selected from the group consisting of 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, and 36 nucleotide monomers; (c) the second strand has a length of any number of nucleotide monomers less than that of the first strand, provided that the second strand can form a duplex with the first strand; and / or (d) at least one of the first and last bases of the second strand is complementary to a nucleobase in the first strand; 14. The asdRNA molecule of claim 13.

15. the second strand is a) 6 to 36 nucleotide monomers; b) 6 to 32 nucleotide monomers; c) 8 to 25 nucleotide monomers and d) 8-23 nucleotide monomers 15. The asdRNA molecule according to any one of claims 13 to 14, having a length selected from the group consisting of:

16. the two ends of the first strand are a) a 3'-overhang and a 5'-overhang; b) a 3'-overhang and a blunt end at the 5' end; c) a 5'-overhang and a blunt end at the 3' end; d) a 3' overhang and a 5' recessed end, and e) 3' recessed end and 5' overhang 16. The asdRNA molecule of any one of claims 1 to 15, selected from the group consisting of:

17. the 3'-overhang of the first strand a) 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, or 30 nucleotide monomers; b) 1 to 15 nucleotide monomers; c) 1 to 10 nucleotide monomers; d) 1 to 8 nucleotide monomers, and e) 1 to 5 nucleotide monomers 17. The asdRNA molecule of claim 16, having a length selected from the group consisting of:

18. the 5'-overhang of the first strand is a) 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, or 30 nucleotide monomers; b) 1 to 15 nucleotide monomers; c) 1 to 10 nucleotide monomers; d) 1 to 8 nucleotide monomers, and e) 1 to 5 nucleotide monomers 17. The asdRNA molecule of claim 16, having a length selected from the group consisting of:

19. (a) the first strand has a 3'-overhang of 1 to 15 nucleotide monomers and a 5'-overhang of 1 to 15 nucleotide monomers; (b) the first strand has a 3'-overhang of 1 to 28 nucleotide monomers and a 5' blunt end or a 5' recessed end; and / or (c) the first strand has a 5'-overhang of 1 to 28 nucleotide monomers and a 3' blunt end or a 3' recessed end; 17. The asdRNA molecule of claim 16.

20. 20. The asdRNA molecule of any one of claims 1 to 19, wherein at least one nucleotide monomer in the first strand and / or the second strand is a modified nucleotide or nucleotide analogue.

21. 21. The asdRNA molecule of claim 20, wherein the modified nucleotide or nucleotide analog is a sugar-, backbone-, and / or base-modified nucleotide.

22. 22. The asdRNA molecule of claim 21, wherein the backbone-modified nucleotide has a modified internucleoside linkage, wherein the internucleoside linkage is modified to include at least one of a nitrogen or sulfur heteroatom, and wherein the modified internucleoside linkage is selected from the group consisting of a phosphorothioate (P=S) group, a phosphotriester, a methylphosphonate, and a phosphoramidate.

23. 21. The asdRNA molecule of claim 20, wherein the first strand and / or the second strand comprises at least one modified internucleoside linkage, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage, and wherein each internucleoside linkage of the first strand and / or the second strand is a phosphorothioate internucleoside linkage.

24. the modified nucleotide or nucleotide analog comprises a modified sugar moiety; (a) the 2' position of the modified sugar moiety is selected from the group consisting of OR, R, halo, SH, SR, NH ２ , N.H.R., N.R. ２ and CN, wherein each R is independently selected from the group consisting of C １ ~C ６ alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I; (b) the 2'-position of the modified sugar moiety is selected from the group consisting of allyl, amino, azido, thio, O-allyl, O-C １ ~C １０ Alkyl, OCF ３ , OCH ２ F, O(CH2) ２ SCH ３ , O(CH ２ ) ２ -O-N (Rm) (Rn), O-CH ２ —C(═O)—N(Rm)(Rn), and O—CH ２ -C(=O)-N(Rl)-(CH ２ ) ２ -N(Rm)(Rn), where R ｌ , R ｍ and R ｎ Each of is independently H or substituted or unsubstituted C １ ~C １０ is alkyl; (c) the modified sugar moiety is 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH ３ , 2'-OCH ２ CH ３ , 2'-OCH ２ CH ２ F and 2'-O(CH2) ２ OCH ３ The substituents are selected from the group consisting of: (d) the modified sugar moiety is 4'-(CH ２ )-O-2'(LNA);4'-(CH ２ )-S-2;4'-(CH ２ )2-O-2'(ENA);4'-CH(CH ３ )-O-2'(cEt) and 4'-CH(CH ２ OCH ３ )-O-2', 4'-C(CH ３ ) (CH ３ )-O-2', 4'-CH ２ -N(OCH ３ )-2',4'-CH ２ -O-N(CH ３ )-2',4'-CH ２ -N(R)-O-2' (wherein R is H, C １ ~C １２ alkyl, or a protecting group), 4'-CH ２ -C(H)(CH ３ )-2', and 4'-CH ２ -C-(=CH ２ )-2'; and / or (e) the modified sugar moiety is a 2'-O-methoxyethyl modified sugar (MOE), 4'-(CH ２ )-O-2' bicyclic sugar (LNA), 2'-deoxy-2'-fluoroarabinose (FANA), and methyl(methyleneoxy) (4'-CH(CH ３ )-O-2) bicyclic sugars (cEt), 21. The asdRNA molecule of claim 20.

25. 20. The asdRNA molecule of any one of claims 1 to 19, wherein the ISD comprises at least one modified nucleotide or nucleotide analog having a modified sugar moiety, wherein the modified sugar moiety is 2'-deoxy-2'-fluoroarabinose (FANA).

26. the modified nucleotide or nucleotide analog comprises a modified nucleobase; (a) the modified nucleobase is 5-methylcytosine (5-Me-C), inosine bases, tritylated bases, 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, 1-methyl-pseudo-uracil, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH ３ ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, and 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine; (b) the modified nucleobase is 5-methylcytosine, and / or (c) each cytosine base is a 5-methylcytosine; 21. The asdRNA molecule of claim 20.

27. 27. The asdRNA molecule of any one of claims 1 to 26, wherein the asdRNA is used to modulate gene expression or function in a cell, wherein the cell is a eukaryotic cell, and wherein the eukaryotic cell is a mammalian cell.

28. 2. The asdRNA molecule of claim 1, wherein the targeted RNA is either an mRNA, a pre-mRNA, an mt-RNA or a non-coding RNA, and such RNA encodes a protein involved in a disease or regulates part of a biological pathway involved in a disease.

29. The target RNA is a) mRNA, pre-mRNA or mt-RNA of a gene involved in a human or animal disease or condition; b) mRNA or pre-mRNA of a gene of a pathogenic microorganism; c) viral RNA; d) lncRNA; e) miRNAs, and f) RNA involved in a disease or disorder selected from the group consisting of an autoimmune disease, an inflammatory disease, a degenerative disease, an infectious disease, a proliferative disease, a metabolic disease, an immune-mediated disorder, an allergic disease, a dermatological disease, a malignant disease, a gastrointestinal disorder, a respiratory disorder, a cardiovascular disorder, a nephrological disorder, a rheumatoid disorder, a neurological disorder, an endocrine disorder, and an aging-related disorder.

2. The asdRNA molecule of claim 1, selected from the group consisting of:

30. 30. The asdRNA molecule of any one of claims 1 to 29, wherein the first strand and / or the second strand is conjugated to a ligand or moiety.

31. 31. The asdRNA molecule of claim 30, wherein the ligand or moiety is selected from the group consisting of a peptide, an antibody, a polymer, a polysaccharide, a lipid, a hydrophobic moiety or molecule, a cationic moiety or molecule, a lipophilic compound or moiety, an oligonucleotide, cholesterol, GalNAc, and an aptamer.

32. 32. A pharmaceutical composition comprising any of the asdRNA molecules of claims 1 to 31 as an active agent and a pharmaceutically acceptable excipient, carrier or diluent.

33. 33. The pharmaceutical composition of claim 32, wherein the carrier is selected from the group consisting of a pharmaceutical carrier, a positively charged carrier, a lipid nanoparticle, a liposome, a protein carrier, a hydrophobic moiety or molecule, a cationic moiety or molecule, GalNAc, a polysaccharide polymer, a nanoparticle, a nanoemulsion, cholesterol, a lipid, a lipophilic compound or moiety, and a lipid.

34. 34. A method for treating or preventing a disease or condition, comprising administering a therapeutically effective amount of an asdRNA molecule described in any one of claims 1 to 31 or a pharmaceutical composition described in any one of claims 32 or 33 to a subject in need thereof.

35. 35. The method of claim 34, wherein the disease or condition is selected from the group consisting of cancer, an autoimmune disease, an inflammatory disease, a degenerative disease, an infectious disease, a proliferative disease, a metabolic disease, an immune-mediated disorder, an allergic disease, a dermatological disease, a malignant disease, a gastrointestinal disorder, a hepatic disorder, a respiratory disorder, a cardiovascular disorder, a dermatological disorder, a nephrological disorder, a rheumatoid disorder, a neurological disorder, a psychiatric disorder, an endocrine disorder, and an aging-related disorder or disease.

36. 36. The method of claim 35, wherein the asdRNA molecule or pharmaceutical composition is administered by a route selected from the group consisting of intravenous injection (iv), subcutaneous injection (sc), orally (po), intramuscular (im) injection, oral administration, inhalation, topical, intrathecal, and other localized administration.

37. 34. A method for modulating gene expression or gene function in a eukaryotic cell, comprising contacting the cell with an effective amount of an asdRNA molecule according to any one of claims 1 to 31 or a pharmaceutical composition according to any one of claims 32 or 33.

38. 1. An asymmetric short double-stranded RNA (asdRNA) molecule comprising a first strand and a second strand, each strand comprising linked ribonucleotide monomers, wherein at least one deoxyribonucleotide monomer interspersed segment (ISD) is located within the first strand, the ribonucleotide monomers are selected from the group consisting of naturally occurring ribonucleotides, analogs thereof, and modified ribonucleotides; and the deoxyribonucleotide monomers are selected from the group consisting of naturally occurring deoxyribonucleotides, analogs thereof, and modified deoxyribonucleotides; at least one ISD comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive deoxyribonucleotide monomers; the second strand is shorter than the first strand by a number of monomers selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 monomers; the first strand is substantially complementary to a segment of an RNA targeted by at least one targeting region, the first strand consisting of 10 to 36 (inclusive) nucleoside monomers linked between adjacent monomers by linkages selected from the group consisting of phosphorothioate linkages, phosphodiester linkages, or mixtures of phosphorothioate and phosphodiester linkages; the second strand is substantially complementary to the first strand and forms at least one double-stranded region with the first strand, the second strand consisting of 8 to 32 (inclusive) nucleoside monomers joined between adjacent monomers by linkages selected from the group consisting of phosphorothioate linkages, phosphodiester linkages, or mixtures of phosphorothioate and phosphodiester linkages; the total number of deoxyribonucleotide monomers is less than or equal to the total number of ribonucleotide monomers in the asdRNA molecule; asdRNA molecule.

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