Asymmetric short double-stranded RNA (Ribonucleic Acid) with deoxyribonucleotide spacer fragment as gene silencing technology and application of asymmetric short double-stranded RNA

Abstract

A novel gene silencing technique for modulating target nucleic acids and / or proteins in cells, tissues, organisms and animals is disclosed. The new technology provides compositions for gene targeting or gene silencing applications, including prevention and treatment of human diseases. The composition comprises an asymmetric, short, duplex RNA molecule in which the sense strand is shorter than the antisense strand. The duplex RNA molecule further comprises at least one deoxynucleotide monomer spacer fragment. The invention further provides methods of using the compositions to modulate the expression or function of a target gene, or for treating or preventing disease and for other medical or biological applications.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefits of U.S. Provisional Patent Application No. 63 / 431,154, filed on December 8, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to a novel gene silencing technology based on asymmetric short double-stranded RNA with deoxyribonucleotide spacers, and related compositions and methods, which can be used for biological or medical research, treatment and prevention of diseases, and gene silencing applications in other biological fields. Background Art

[0003] Modern medical therapies rely on two fundamental technologies: small molecule chemistry and protein / antibody technology. However, only approximately 10% of the targets identified by genomic and biomedical research can be addressed using these two foundational technologies. Oligonucleotides hold the potential to address numerous targets, including those undruggable using small molecule chemistry and protein / antibody technology. Over 40 years of research have led to the development of antisense oligonucleotide (ASO) and small interfering RNA (siRNA) technologies (Cy A. Stein et al., 2017). However, despite these 40 years of research, significant druggability issues have hindered the development of ASO and siRNA technologies into mainstream therapeutic platforms, with the exception of a few orphan clinical indications. These druggability issues include, among others: low silencing efficacy, off-target effects, stimulation of unintended immune responses, tissue penetration challenges, and in vivo delivery challenges. Therefore, there is a significant unmet need for developing new technologies to target target genes in a variety of biological and medical applications.

[0004] ASO is a gene silencing technology based on a concept first proposed in 1978 (Zamecnik P.C. et al., 1978). Generally speaking, the principle behind ASO technology is that antisense oligonucleotides hybridize to target nucleic acids and regulate the activity or function of gene expression, such as transcription / post-transcription or translation. The mechanisms of ASOs are broadly categorized as follows: (1) occupancy without promoting RNA degradation, where ASO binding leads to translational arrest, splicing inhibition, or induction of alternative splice variants, or (2) occupancy-induced destabilization, where ASO binding promotes RNA degradation by endogenous enzymes, such as ribonuclease H1 (RNase H1); and (3) translational regulation: ASOs can block upstream open reading frames (uORFs) or other inhibitory or regulatory elements in the 5'UTR region, thereby increasing or modulating translation efficiency (Stanley T. Crooke et al., 2008; C. Frank Bennett, 2010; Richard G. Lee, 2013; Stanley T. Crooke, 2017). ASOs are single-stranded deoxyribonucleotide sequences that can bind to target RNAs through base pairing. After 40 years of research, ASO technology has been improved through various chemical modifications of single-stranded oligonucleotides, such as phosphorothioate substitutions or other modified nucleotides (see Iwamoto Net al 2017, Crooke ST, 2017; Crooke ST et al., 2018; US Pat. Nos. 7919472 and 9045754).

[0005] Short double-stranded RNAs (dsRNAs) trigger the loss of homologous RNA sequences through the RNAi mechanism, a mechanism first observed in plants and confirmed in the nematode Caenorhabditis elegans (A. Fire et al., 1998). This mechanism involves the degradation of long dsRNAs into short interfering duplex RNAs (siRNAs), which interact with the multiprotein RNA-Induced Silencing Complex (RISC). Within RISC, the siRNA unwinds, the sense strand is discarded, and the antisense, or guide, strand binds to the RISC endonuclease AGO2, which then cleaves the target RNA (deFougerolles et al., 2007; Ryszard and Kole, 2016). RNAi is a sequence-specific posttranscriptional gene silencing process triggered by short double-stranded RNA in the cytoplasm and can therefore be used to silence mRNAs in the cytoplasm. In mammalian cells, synthetic siRNA or asymmetric short interfering RNAs (aiRNA or asymmetric siRNA) can be used to induce gene silencing via the RISC-dependent RNAi mechanism (see Elbashir SM et al., 2001; Sun X et al., 2008; US Pat. Nos. 7056704 and 9328345).

[0006] Oligonucleotides, studied for decades, are considered promising candidates for a novel class of therapeutics. However, their limited silencing efficiency, delivery challenges, and dose-dependent side effects (including hybridization-dependent and hybridization-independent toxicities) have hindered the development of these novel therapies (C. FrankBennett, 2010; C. FrankBennett, 2019; Roberts TC et al., 2020; Crooke ST et al., 2018; and Setten RL et al., 2020). Generally speaking, although ASO compounds are less potent than siRNA-based compounds in inducing gene silencing, they possess 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 primarily due to hybridization with non-target genes ("off-target effects") (Jackson et al., 2003; LinX et al., 2005). Hybridization-independent toxicity of oligonucleotides occurs through interactions with proteins: 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 can cause renal tubulopathy and thrombocytopenia (Geary, RS. et al., 2007; Kwoh JT, 2008). Clinically, the primary tolerability and safety issues associated with first-generation PS antisense oligodeoxynucleotides and second-generation 2'-MOE-modified antisense oligonucleotides have been demonstrated 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 are generally still far less effective than siRNAs and have been shown to exhibit typical dose-dependent toxicity (Kendall S. Frazier, 2015). Over the past 40 years, efforts have been made to mitigate the dose-dependent toxicity of oligonucleotides through various chemical modifications to overcome the limited efficacy and associated safety issues of ASOs (Iwamoto 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 believed 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). In addition, typical 21nt / 19bp siRNA duplexes are not efficient in cell and tissue penetration and require extensive chemical modifications to enhance siRNA stability and other drug properties. To overcome the off-target effects and other off-target mechanisms mediated by the sense strand of symmetrical siRNA, asymmetric siRNAs (or aiRNAs) have been designed (see Sun X et al., 2008; Grimm D, 2009; Selbly CR et al., 2010; and PCT application WO2009029688).

[0008] In summary, after more than 40 years of innovation in ASO technology and over 20 years of RNAi-based research, the successful development of gene-targeted therapies for nearly 90% of targets associated with human disease remains challenging. Furthermore, currently approved oligonucleotide-based drugs cost over $500,000 per patient per year, making them inadequate for addressing diseases affecting the general population. Therefore, new technologies are urgently needed to overcome these challenges.

[0009] The citation of references herein is not to be construed as an admission that they are prior art to the claimed invention. SUMMARY OF THE INVENTION

[0010] The present invention is based on the unexpected discovery of effective gene silencing triggered by asymmetric short duplex ribonucleotides (asdRNA) with an interspersed segment of deoxyribonucleotides (ISD). This novel gene silencing technology is achieved by asdRNA with one or more deoxyribonucleotide spacers. The asdRNA employs a short duplex molecule composed of linked nucleotide monomers, wherein each nucleotide monomer is selected from the group consisting of naturally occurring nucleotides, their analogs, and modified nucleotides (hereinafter collectively referred to as "nucleotide monomers"). In other words, the nucleotide monomers used in one embodiment of the present invention comprise "ribonucleotide monomers," wherein the "ribonucleotide monomers" are selected from the group consisting of naturally occurring ribonucleotides, their analogs, and modified ribonucleotides. Furthermore, by incorporating one or more deoxyribonucleotide monomer spacers, the gene silencing function of the asdRNA can be significantly achieved or enhanced. "Deoxyribonucleotide monomers" may be selected from the group consisting of naturally occurring deoxyribonucleotides, analogs thereof, and modified deoxyribonucleotides.

[0011] In the present invention, at least 50% of the nucleotide monomers in the asdRNA molecule of the invention are ribonucleotide monomers, and thus 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 invention are further interrupted by deoxyribonucleotide monomers, forming at least one intervening deoxyribonucleotide monomer segment (ISD).

[0012] In one embodiment, the powerful gene silencing effect of the novel platform technology based on asdRNA included in the present disclosure and other advantages disclosed below are realized by oligonucleotide monomer sense strand and oligonucleotide monomer antisense strand, wherein the oligonucleotide monomer antisense strand is substantially complementary to the target ribonucleotide sequence. Our data show that the asdRNA molecule of the present invention can cause gene silencing under picomolar (such as 800pM, 500pM, 300pM, 200pM, 100pM or even lower concentration) concentration because of its unique, novel composition, which is stronger than existing gene silencing technology efficacy, and therefore can reduce dose-dependent toxicity. The asdRNA molecule of the present invention also has at least one following advantage that is superior to existing gene silencing technology, including that gene silencing can be realized in cytoplasm and nucleus and mitochondria etc. (by contrast, gene silencing based on siRNA / aiRNA only occurs in cytoplasm); Reduce off-target effect; Eliminate or reduce the undesirable interference to endogenous microRNA function present in siRNA; Better tissue permeability; Better stability; Low synthesis cost and improved pharmaceutical properties. Therefore, asdRNA molecule of the present invention has huge potential to solving the various challenges faced by ASO, siRNA / aiRNA and other existing gene silencing technologies. In addition, the asdRNA molecule of the present invention can regulate gene expression activity or function, post-transcriptional stage and / or translation stage, and RNAi can only trigger gene silencing at the post-transcriptional level. In addition, asdRNA can allow more and more extensive chemical modifications, including nucleotide modifications or replacements of non-RNA samples (non-RNAlike). The asdRNA molecule of the present invention can be used for the whole fields of current oligonucleotide application or expected use, including other applications of research, diagnosis, disease prevention and treatment and biological fields, and also includes pesticide and veterinary drug fields.

[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, wherein the first strand and the second strand each comprise a linked ribonucleotide monomer and a deoxyribonucleotide monomer spacer. The ribonucleotide monomers in the asdRNA molecule are selected from the group consisting of: naturally occurring ribonucleotides, their analogs, and modified ribonucleotides; the deoxyribonucleotide monomer spacer in the asdRNA molecule is selected from the group consisting of: naturally occurring deoxynucleotides, their analogs, and modified deoxynucleotides. The asdRNA molecule is an asymmetric short duplex RNA (asdRNA) molecule, wherein the second strand of the asdRNA molecule is shorter than the first strand. Since the first strand is substantially complementary to the target fragment of the target RNA through at least one targeting region, the first strand can be considered an antisense strand or an antisense oligonucleotide. Furthermore, the second strand is substantially complementary to the first strand, forming at least one double-stranded region with the first strand, and the second strand can also be considered a sense strand or a sense oligonucleotide. The asdRNA molecule comprises at least one deoxynucleotide monomer spacer (ISD) having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deoxynucleotide monomers, wherein the ISD can be present in either strand or in both strands. The total number of deoxynucleotide monomers in the asdRNA molecule of the present invention does not exceed the total number of ribonucleotide monomers in any given asdRNA molecule. In one feature, at least a portion of at least one targeting 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 have the same meaning.

[0015] In one feature, an asdRNA containing an ISD has improved gene regulatory or pharmaceutical properties compared to a corresponding (asymmetric) RNA duplex without the ISD. In other words, the asdRNA disclosed herein comprising at least one ISD has improved or more desirable at least one gene regulatory or pharmaceutical property selected from the group consisting of: function in different subcellular locations in addition to the cytoplasm, potential target RNAs of interest, efficacy, potency, off-target effects, speed of onset, durability, economical synthesis, available chemical modifications, nonspecific immune stimulation, stability, and delivery. More specifically, the improved gene regulatory properties or pharmaceutical properties of the asdRNA molecules of the present invention compared to corresponding RNA duplexes refer to, for example, one or more of the following situations: the ability to achieve gene regulatory functions not only in the cytoplasm of cells, but also in the nucleus and / or mitochondria; the ability to target more types of RNA (including not only mRNA, but also pre-mRNA, non-coding RNA, long non-coding RNA and mt-mRNA (mitochondrial messenger RNA)); better efficacy and / or potency; fewer off-target effects; faster onset of action; improved pharmacokinetic properties; longer persistence; less typical toxicity of dose-dependent resistance; avoidance of nonspecific interferon-like reactions; and lower manufacturing costs; tolerance / possession of more chemical modifications (including non-RNA-like nucleotide modifications or substitutions), better stability and better delivery. The corresponding (asymmetric) RNA duplex refers to an (asymmetric) short double-stranded RNA molecule that does not contain the ISD of the present application, wherein its antisense strand targets the same or substantially the same sequence as at least one targeting region in the first strand of the asdRNA molecule. More specifically, asdRNA with an ISD can also be used to target or silence RNA in the cell nucleus, such as pre-mRNA, non-coding RNA, and long non-coding RNA, and can also be used to target or silence RNA in mitochondria, such as mt-mRNA (mitochondrial messenger RNA), while RNAi technologies with (asymmetric) short double-stranded RNA structures (such as aiRNA and siRNA) can only function in the cytoplasm. Therefore, compared with existing gene silencing technologies, especially RISC-dependent RNAi gene silencing technologies, asdRNA with an ISD can be used to target more target RNAs, pathogenic genes, and have broader application prospects. In another feature, the asdRNA molecules of the present invention have better gene regulatory or pharmaceutical properties than corresponding single-stranded antisense oligonucleotides (ASOs). In other words, at least one gene regulatory property or pharmaceutical property of the asdRNA is better or more ideal than that of the corresponding ASO; the property is selected from the following group: efficacy, potency, speed of onset, durability, synthetic economy, off-target effects, nonspecific 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 targeting region in the first strand of the asdRNA molecule.

[0016] The present invention provides compositions for regulating gene expression or function in eukaryotic cells, wherein the asdRNA is contacted with the cells or administered to a subject.

[0017] In one feature, the first strand of an asdRNA molecule of the invention comprises at least one ISD, and / or the second strand thereof may comprise at least one ISD. In one embodiment, the first strand of the asdRNA molecule comprises at least one ISD, and the second strand also comprises at least one ISD. In one feature, at least one ISD is located in at least one targeting region of the first strand, and at least one ISD is located in at least one double-stranded region of the second strand. In one embodiment, the first strand of the asdRNA molecule comprises at least one ISD, and the second strand is composed of ribonucleotide monomers.

[0018] In one feature, each ISD is independently composed of a single deoxynucleomonomer or comprises at least 2, 3, 4, 5, or more consecutive deoxynucleomonomers. In one embodiment, at least one ISD comprises at least 4 consecutive deoxynucleomonomers. In another feature, an ISD comprises at least 2 deoxynucleomonomers, wherein the deoxynucleomonomers are consecutive or separated by incorporation of at least one (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) different monomer species. In one feature, an ISD is separated by incorporation of at least 2 (2, 3, 4, 5, 6, 7, 8, 9, 10, or more) different monomer species. In another feature, the total number of deoxynucleomonomers in all ISDs in the first strand is at least 2.

[0019] In one embodiment, at least one ISD is distributed throughout the first strand. In one feature, at least one ISD is distributed 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 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive deoxynucleomonomers. In various embodiments, at least one ISD within the first strand comprises at least 4 consecutive deoxynucleomonomers. In various embodiments, at least one ISD within the targeting region of the first strand comprises at least 4 consecutive deoxynucleomonomers. In one embodiment, there is only one ISD within the first strand that comprises at least 4 consecutive deoxynucleomonomers. In another embodiment, there are two or more ISDs within the first strand, wherein each ISD independently consists of a single deoxynucleomonomer, or comprises at least 2, 3, 4, 5, or more consecutive deoxynucleomonomers. In another embodiment, there are two or more ISDs within the first strand, wherein one ISD comprises at least 4 consecutive deoxynucleomonomers and the other ISDs each independently consist of a single deoxynucleomonomer, or comprise at least 2, 3, 4, 5, or more consecutive deoxynucleomonomers.

[0020] In one embodiment, at least one ISD is distributed in the second strand. In one feature, at least one ISD is distributed in at least one double-stranded region of the second strand. In various embodiments, at least one ISD in the second strand comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive deoxynucleoside monomers. In another feature, the ISD in the second strand comprises at least 2 deoxynucleoside monomers, wherein the deoxynucleoside monomers are consecutive or separated by incorporation of at least one (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) monomer of a different species.

[0021] In some embodiments, the ISD is located in the more central portion of the first strand (at least 1, 2, 3, 4, or 5 nucleotides from either end of the strand, i.e., the second position or more central position from the end of the strand). In certain embodiments, the ISD can be located at any position in the second strand. In some embodiments, the ISD is located in the more central portion of the second strand (at least 1, 2, 3, 4, or 5 nucleotides from either end of the strand, i.e., the second position or more central position from the end of the strand). In certain embodiments, at least one end of the first and / or second strand (i.e., the first nucleomonomer from the 3' end, the 5' end, or both) is not a deoxynucleomonomer.

[0022] In one feature, the first strand comprises a plurality of linked nucleotide monomers to form a nucleobase sequence, and the first strand is at least 70%, 80%, 85%, 90%, 95%, or completely complementary to a target segment of an RNA of a target gene. In certain embodiments, the target RNA is selected from mRNA, pre-mRNA, mt-mRNA, and non-coding RNA, wherein the RNA encodes a protein associated with a disease or regulates a portion of a biological pathway associated with a disease, such as 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 the equivalent thereof, or a range of lengths encompassed by any two of the foregoing values ​​(both endpoints of the range are inclusive). For example, some ranges of lengths for the first strand (antisense strand) include: (a) 8-33 nucleotide monomers; (b) 10-30 nucleotide monomers; (c) 10-29 nucleotide monomers; (d) 12-29 nucleotide monomers; (e) 12-28 nucleotide monomers; (f) 12-26 nucleotide monomers; (g) 12-25 nucleotide monomers; (h) 13-25 nucleotide monomers; (i) 13-24 nucleotide monomers; (j) 13-23 nucleotide monomers; (k) 15-23 nucleotide monomers; (l) 8-50 nucleotide monomers; (m) 10-36 nucleotide monomers; (n) 12-36 nucleotide monomers; (o) 12-32 nucleotide monomers; (p) 14-36 nucleotide monomers; and (q) at least 8 nucleotide monomers. In certain embodiments, when the backbone of the first strand is 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 nucleomonomers in length, or the equivalent thereof, the at least one ISD can be located anywhere in the first strand and, if present in the second strand, anywhere in the second strand.

[0024] In one feature, the second strand comprises a plurality of linked nucleotide monomers to form a nucleobase sequence, and the second strand is at least 70%, 75%, 80%, 85%, 90%, 95%, or completely complementary to at least one linked region of the first strand. In some embodiments, the sense strand is completely complementary to at least one linked region of the first strand and forms 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 strand or the antisense strand and forms at least one double-stranded region with 1, 2, 3, or more mismatches. In one feature, the mismatched monomers in the sense strand have nucleobases selected from the group consisting of A, G, C, U, and T or selected from modified nucleobases. In a specific embodiment, 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 base and the last base of the second strand are complementary to a nucleobase in the first strand.

[0025] In one feature, the second strand has a backbone length that is shorter than the first strand by at least 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 nucleomonomers, or the equivalent thereof, or a range of lengths encompassed by any two of the foregoing values ​​(both endpoints of the range are inclusive). For example, in certain embodiments, some ranges of lengths of the second strand / sense strand include: (a) 8-32 nucleotide monomers; (b) 8-30 nucleotide monomers; (c) 8-29 nucleotide monomers; (d) 9-29 nucleotide monomers; (e) 9-26 nucleotide monomers; (f) 9-25 nucleotide monomers; (g) 10-29 nucleotide monomers; (h) 10-28 nucleotide monomers; (i) 10-26 nucleotide monomers; (j) 10-25 nucleotide monomers; (k) 11-24 nucleotide monomers; (l) 11-23 nucleotide monomers. In some embodiments, the backbone length of the second strand can have any number of nucleotide monomers less than the length of the first strand, provided that the second strand is capable of forming a thermodynamically stable duplex with the first strand.

[0027] In one feature, the two ends of the first strand are one of the following configurations: a 3' overhang and a 5' overhang, a 3' overhang and a 5' blunt end, a 5' overhang and a 3' blunt 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 encompassed by any two of the above values. In various embodiments, the 3' overhang of the first strand has a length of 1-15, 1-10, 1-8, or 1-5 nucleotide monomers (both ranges are inclusive). 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 encompassed by any two of the foregoing values. In various embodiments, the 5' overhang of the first strand has a length of 1-15, 1-10, 1-9, 1-8, or 1-5 nucleotide monomers.

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

[0029] In one feature, the two ends of the second strand are 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 1-15, 1-10, 1-9, 1-8, or 1-5 nucleotide monomers (both end points of the range are included). 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 1-15, 1-10, 1-9, 1-8, or 1-5 nucleotide monomers (both ranges are inclusive).

[0030] In one feature of the asdRNA molecules of the invention, at least one nucleotide monomer in the first and / or second strand is a modified nucleotide or nucleotide analog, e.g., a sugar-modified, backbone-modified, and / or base-modified nucleotide. In one embodiment, the backbone-modified nucleotide has a modification in at least the internucleoside linkage, e.g., including at least one of a nitrogen heteroatom or a sulfur heteroatom. In certain embodiments, the modified internucleoside linkage is or comprises a phosphorothioate group (P=S), a phosphotriester, a methylphosphonate, or a phosphoramidate.

[0031] In certain embodiments, the first chain and / or the second chain comprises at least one modified internucleoside bond, wherein the modified internucleoside bond is a phosphorothioate internucleoside bond. In some embodiments, the bond between each internucleoside of the first chain and / or the second chain is a phosphorothioate internucleoside bond. In various embodiments, the bond between the nucleosides of the first chain and / or the second chain is a mixture of a phosphorothioate bond and a phosphodiester bond. In certain embodiments, the bond between each nucleoside of the first chain is a modified internucleoside bond, and the bond between each nucleoside of the second chain is a naturally occurring internucleoside bond.

[0032] In one feature, the first and / or second strands of the molecules of the invention comprise at least one modified nucleotide or nucleotide analog, wherein the modified nucleotide or nucleotide analog comprises a modified sugar moiety. In certain embodiments, the 2' position of the modified sugar moiety is substituted by a group selected from the group consisting of OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein each R is independently C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I. In some embodiments, the 2' position of the modified sugar moiety is substituted by a group selected from the group consisting of allyl, amino, azido, thio, O-allyl, O-C1-C6 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 ), where each R1, R m and R n are independently H or substituted or unsubstituted C1-C 10 In some embodiments, the modified sugar moiety has a substituent group selected from the group consisting of 5'-vinyl, 5'methyl (R or S), 4'-S, 2'-F, 2'-OCH3, 2'-OCH2CH3, 2'-OCH2CH2F, 2'-O-aminopropylated (2'-AP), and 2'-O(CH2)2OCH3. In some embodiments, the modified sugar moiety is substituted with a bicyclic sugar selected from the group consisting 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(CH2OCH3)—O-2′, 4′-C(CH3)(CH3)—O-2′, 4′-CH2—N(OCH3)-2′, 4′-CH2—O—N(CH3)-2′, 4′-CH2—N(R)—O-2′ (where R is H, C1-C 12 In some embodiments, the modified sugar moiety is selected from the group consisting of 2'-O-methoxyethyl modified sugar (MOE), 4'-(CH2)-O-2' bicyclic sugar (LNA), 2'-deoxy-2'-fluoroarabinose (2'-F arabinose, FANA), and methyl(methyleneoxy)(4'-CH(CH3)-O-2) bicyclic sugar (cEt).

[0033] In a specific embodiment, 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 another specific embodiment, the ISD may comprise at least one CpG motif, wherein the CpG motif can be recognized by a pattern recognition receptor (PRR), such as a Toll-like receptor.

[0035] In one feature of the asdRNA molecules of the invention, the sugar portion 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).

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

[0037] In another feature, the first and / or second chains of the molecules of the present invention include at least one nucleotide monomer, wherein the nucleotide monomer comprises a modified nucleobase. In some embodiments, the modified nucleobase is selected from the following group: 5-methylcytosine (5-Me-C), hypoxanthine nucleoside base, tritylated base, 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 other alkynyl derivatives of cytosine and pyrimidine bases, 6-azouracil , cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 1-methyl-pseudouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy 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 a specific embodiment, the modified nucleobase is 5-methylcytosine. In one embodiment, each cytosine base in the molecules of the present invention is 5-methylcytosine. In one embodiment, each uridine base in the ribonucleotide monomers of the asdRNA molecule of the invention is 5-methyluridine.

[0038] In one feature, the first and / or second chains of the molecules of the invention are conjugated to a ligand or moiety. In one embodiment, the ligand or moiety is selected from the group consisting of a polypeptide, 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 a nucleic acid aptamer.

[0039] In one feature of the invention, asdRNA molecules are used to modulate gene expression or function in a cell (eg, a eukaryotic cell, such as a mammalian cell).

[0040] In certain embodiments, the target RNA, which determines at least a portion of the nucleotide monomer sequence of the asdRNA molecule according to the principles of the present invention, is selected from mRNA, pre-mRNA, mt-mRNA, or non-coding RNA. In one feature, these target RNAs either encode a protein associated with a disease or regulate a portion of a biological pathway associated with a disease. In various embodiments, such target RNAs can be, but are not limited to, selected from: mRNA, pre-mRNA, mt-mRNA, non-coding RNA, or lncRNA of a gene associated with a disease or condition in humans or animals; mRNA or pre-mRNA of a pathogenic microbial gene; viral RNA; and RNA associated with 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, skin diseases, malignancies, gastrointestinal diseases, respiratory diseases, cardiovascular diseases, kidney diseases, rheumatoid diseases, neurological diseases, endocrine disorders, and aging-related diseases or disorders.

[0041] In one embodiment, the present invention provides an asymmetric short double-stranded RNA (asdRNA) molecule comprising a first strand and a second strand, each strand comprising linked ribonucleotide monomers, wherein the ribonucleotide monomers are selected from the group consisting of naturally occurring ribonucleotides, analogs thereof, and modified ribonucleotides, and at least one spacer segment of deoxyribonucleotide monomers (ISD), wherein: (a) the first strand is longer than the second strand by at least a number of monomers selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and (b) the first strand is substantially complementary to a target segment of a target RNA through at least one targeting region, wherein the first strand is composed of 10-36 nucleoside monomers (both ends of the range are inclusive) linked by bonds selected from the group consisting of phosphorothioate bonds, phosphodiester bonds, and mixtures of phosphorothioate and phosphodiester bonds between adjacent monomers. (c) the second strand is substantially complementary to the first strand and forms at least one double-stranded region with the first strand, wherein the second strand is composed of 8-32 (both ends of the range are inclusive) nucleoside monomers connected by bonds selected from the group consisting of phosphorothioate bonds, phosphodiester bonds, and mixtures of phosphorothioate and phosphodiester bonds between adjacent monomers; (d) at least one ISD is linked to at least one ribonucleotide monomer, wherein the ribonucleotide monomer is selected from the group consisting of ribonucleotides, analogs thereof, and modified ribonucleotides; (e) the ISD in the asdRNA molecule includes at least one deoxynucleotide monomer, wherein the deoxynucleotide monomer is selected from the group consisting of deoxynucleotides, analogs thereof, and modified deoxynucleotides; and (f) the total number of deoxynucleotide monomers in the asdRNA molecule does not exceed the total number of ribonucleotide monomers therein. In one feature, the asdRNA molecule is used to modulate the expression or function of a target gene in a cell (e.g., a eukaryotic cell, such as a mammalian cell). In further features, the asdRNA molecule silences the expression of the target gene in the cell more strongly or more efficiently than a corresponding ASO. In a further feature, the asdRNA molecule can not only realize gene regulatory functions in the cytoplasm of the cell, but also realize gene regulatory functions in the nucleus and / or mitochondria, and thus can target the RNA in the nucleus and / or mitochondria in the cell. In a further feature, the asdRNA molecule makes the silencing effect of target gene expression in the cell stronger or more effective than the corresponding (asymmetric) RNA duplex. Although the mechanism of action of the asdRNA in the present invention is unclear, current studies have shown that when Ago2 is knocked out, the gene silencing efficacy of the asdRNA in the present invention is not affected, which indicates that the asdRNA in the present invention may not work through a RISC-dependent mechanism. Therefore, asdRNA can be used to target target genes or target sequences that are resistant 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 therefor. Examples of such carriers include, but are not limited to, drug carriers, positively charged carriers, liposomes, lipid nanoparticles, protein carriers, hydrophobic moieties or molecules, cationic moieties or molecules, GalNAc, polysaccharide polymers, nanoparticles, nanoemulsions, cholesterol, lipids, lipophilic compounds or moieties, and lipids.

[0043] In a third aspect, the present invention provides a method for treating or preventing a disease or condition using the composition of the first aspect or the pharmaceutical composition of the second aspect, by administering a therapeutically effective amount of an asdRNA molecule of the present invention or a pharmaceutical composition comprising the asdRNA molecule. The administration method is selected from the group consisting of intravenous (iv), subcutaneous (sc), oral (po), intramuscular (im), oral administration, inhalation, topical, intrathecal, and other administration methods.

[0044] In one feature, the disease or condition being treated prophylactically or therapeutically is selected from the group consisting of cancer, autoimmune diseases, inflammatory diseases, degenerative diseases, infectious diseases, proliferative diseases, metabolic diseases, immune-mediated disorders, allergic diseases, skin diseases, malignancies, gastrointestinal diseases, liver diseases, respiratory diseases, cardiovascular diseases, skin diseases, kidney diseases, rheumatoid diseases, neurological diseases, psychiatric diseases, endocrine disorders, and diseases or disorders associated with aging.

[0045] In a fourth aspect, the present invention provides a method for regulating or modulating gene expression or gene function in eukaryotic cells using the composition of the first aspect or the pharmaceutical composition of the second aspect. The method comprises the following steps: contacting the cells with an effective amount of any asdRNA molecule of the present invention or a pharmaceutical composition comprising the asdRNA molecule.

[0046] In one embodiment, the contacting step comprises the step of introducing a composition comprising the asdRNA molecule into a target cell or organism in culture where selective gene silencing can occur. In another embodiment, the introducing step is selected from the group consisting of simple mixing, transfection, lipofection, electroporation, infection, injection, oral administration, intravenous (iv), subcutaneous (sc), oral (po), intramuscular (im), inhalation, topical, intrathecal, and other site administration. In another embodiment, the introducing step comprises the use of a pharmaceutically acceptable excipient, carrier, or diluent, wherein the pharmaceutically acceptable excipient, carrier, or diluent is selected from the group consisting of a drug 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.

[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 a non-coding RNA such as microRNA and lncRNA.

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

[0049] In another aspect, the present invention provides an asymmetric oligoduplex comprising (a) one or more ribonucleosides, analogs thereof, or modified ribonucleosides, and (b) one or more ISDs linked to an antisense sequence to form an antisense strand at least 8 nucleobases in length, wherein the ISDs comprise deoxyribonucleosides, analogs thereof, or modified deoxyribonucleosides. The antisense sequence is at least 70% complementary to a target sequence.

[0050] Other features and advantages of the present invention are apparent from the additional description (including different embodiments) provided herein. The embodiments provided illustrate different components and methods useful in implementing the present invention. The embodiments do not limit the claimed invention. According to the content of this disclosure, those skilled in the art can confirm and adopt other components and methods useful for implementing the present invention. Several embodiments have been shown and described, but any modification can be made without departing from the spirit and scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Shown are representative target genes, representative target sequences used in the Examples, and exemplary sequences of corresponding antisense molecules that can be used to silence the target genes.

[0052] Figure 2A Shown are exemplary structures of some embodiments of asdRNA, wherein the antisense strand (first strand) of the asdRNA has at least one deoxyribonucleotide monomer spacer (ISD), and the sense strand (second strand) is pure RNA. In each duplex described herein, the sense strand is listed above the antisense strand. Figure 2B Shows the Figure 2A Exemplary sequences of asdRNAs targeting the APOIII gene are shown. Figure 2C AsdRNA targeting the APOCIII gene ( Figure 2B ) and the corresponding ASO (the corresponding ASO and Figure 2B The relative mRNA levels of APOIIIC genes were measured after HepaRG cells were transfected with 100 pM of asdRNA and the corresponding ASO.

[0053] Figure 3A Shown are exemplary structures of some embodiments of asdRNAs having various ISD motifs in the antisense strand, as well as exemplary sequences of asdRNAs targeting the APOCIII gene. Figure 3A The various ISD motifs in the antisense strand have different numbers of deoxyribonucleotide monomers and different positions of the ISD in the antisense strand. Figure 3B AsdRNAs targeting the APOCIII gene ( Figure 3A ) and the comparison of gene silencing efficacy with the corresponding ASO (each corresponding ASO and Figure 3A The antisense strand sequence of each asdRNA is the same). After transfecting HepaRG cells with 100 pM asdRNA and the corresponding ASO, the relative mRNA level of the APOIIIC gene was detected.

[0054] Figure 4A Shown are exemplary structures of some embodiments of asdRNAs having at least one ISD only in the antisense strand, as well as exemplary sequences of asdRNAs targeting the APOB gene. Figure 4B AsdRNA targeting APOB gene is shown ( Figure 4A The relative mRNA levels of the APOB gene were determined by transfecting HepaRG cells with 5 nM asdRNA.

[0055] Figure 5 Shown are the sequences of exemplary asdDNAs targeting the β-Catenin gene, and the gene silencing efficacies of asdRNAs at concentrations of 100 pM, 200 pM, 1 nM, 3 nM, 10 nM, and 30 nM in DLD1 cells, respectively. Detailed Description of the Invention

[0056] The present invention relates to a novel short duplex RNA gene or RNA regulation / silencing technology. This new technology utilizes asymmetric short duplex RNA (asdRNA) compositions with deoxynucleotide spacers for regulating gene expression or function in vitro and in vivo. The present invention also provides methods for using these compositions to regulate target gene expression or function, or for treating or preventing disease, as well as for other medical and biological applications. These compositions and methods provide high efficacy in regulating gene expression or gene function while also reducing dose-dependent toxicity. 1. Definition

[0057] As used herein, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural forms thereof. 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 defines the range by extending the upper and lower boundaries of these numerical values. Generally speaking, the term "about" is used herein to define the numerical value with a variation of 20%, 10%, 5% or 1% above and below the set value. In some embodiments, the term "about" is used to define the numerical value with a variation of 10% above and below the set value. In some embodiments, the term "about" is used to define the numerical value with a variation of 5% above and below the set value. In some embodiments, the term "about" is used to define the numerical value with a variation of 1% above and below the set value.

[0059] As used herein, the terms "analog" or "analogue" interchangeably refer to functionally or structurally equivalent. For example, nucleoside and nucleotide analogs have been used in the clinical treatment of cancer and viral infections for decades, and researchers and the pharmaceutical industry are constantly synthesizing and evaluating new compounds, 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 core base 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), analogs thereof, and modified nucleosides. Nucleoside monomers can be deoxyribonucleoside monomers or ribonucleoside monomers. For example, a nucleoside monomer can be linked to a phosphate moiety to form a nucleotide monomer.

[0063] As used herein, the term "nucleotide" refers to a nucleoside further comprising a phosphate linking group. Nucleoside monomers include, but are not limited to, naturally occurring nucleotides (e.g., deoxyribonucleotides and ribonucleotides found in DNA and RNA, respectively), analogs thereof, and modified nucleotides. Nucleoside monomers can be deoxyribonucleotide monomers or ribonucleotide monomers. Modified nucleotides can be modified at one or more of the following: a nitrogenous core base moiety, a five-carbon sugar moiety, and a phosphate linking group that causes changes in the internucleoside bond.

[0064] As used herein, the term "oligonucleotide (abbreviated as oligo)" or "oligonucleotide" refers to a compound comprising a plurality of linked nucleoside monomers. In certain embodiments, one or more nucleoside monomers are modified, or one or more internucleoside bonds 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 "duplex RNA" in "short duplex RNA (sdRNA)" or "asymmetric short duplex RNA (asdRNA)" refers to a molecule composed of two nucleotide monomer chains that hybridize to each other to form a duplex oligonucleotide and are contacted with a cell or administered to a subject, wherein the majority, i.e., 50% or more, of the linked nucleotide monomers are ribonucleotide monomers, including modified ribonucleotides.

[0067] As used herein, the term "motif" is a pattern of chemically distinct regions, such as in the antisense strand or the sense strand.

[0068] As used herein, the term "immediately adjacent" refers to the absence of intervening elements between two elements, such as between regions, fragments, nucleotides and / or nucleosides.

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

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

[0071] As used herein, the term "modified oligonucleotide" refers to an oligonucleotide comprising at least one modified nucleotide.

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

[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" refers to a sugar that is naturally present in DNA (2-H) or RNA (2-OH).

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

[0076] As used herein, the term "bicyclic sugar" refers to a furosyl ring modified by bridging two non-bicyclic 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 whose furanose portion includes a bridging group connecting two carbon atoms on the furanose ring to form a bicyclic sugar system.

[0078] As used herein, the term "2'-O-methoxyethyl" (also known as 2'-O-(CH2)2-OCH3 and 2'-O-(2-methoxyethyl)) refers to a furanosyl ring with a 2'-O-methoxyethyl modification of the 2' position. A 2'-O-methoxyethyl-modified sugar is a modified sugar. As used herein, the term "2'-O-methoxyethyl nucleotide" (also known as 2'-MOE RNA) refers to a modified nucleotide comprising a 2'-O-methoxyethyl-modified sugar moiety.

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

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

[0081] As used herein, "RNA-like nucleotide" refers to a modified nucleotide that adopts a Northern configuration when incorporated into an oligonucleotide and functions 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, tricyclic DNA (tcDNA), and RNA surrogates.

[0082] As used herein, "DNA-like nucleotide" refers to a modified nucleotide that functions like DNA when incorporated into an oligonucleotide. DNA-like nucleotides include, but are not limited to, 2'-deoxy-2'-fluoroarabino (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 any of the aforementioned mimics (mimic). As used herein, "lncRNA" and "long non-coding RNA" are transcribed RNA molecules that contain more than 200 nucleotides that do not encode proteins. LncRNA can also undergo common post-transcriptional modifications, including 5'-capping, 3'-polyadenylation and splicing. In general, lncRNA is a diverse class of molecules that play various roles in regulating the function of genes and genomes. For example, it is well known that lncRNAs regulate gene transcription, translation and epigenetic regulation. Examples of IncRNA include, but are not limited to: Kcnqlot1, Xlsirt, Xist, ANRIL, NEAT1, NRON, DANCR, OIP5-AS1, TUG1, CasC7, HOTAIR, and MALAT1. As used herein, "splicing" or "splicing" refers to the natural process of removing unnecessary RNA regions and transforming RNA. An example of regulating RNA target function by asdRNA is the regulation of non-coding RNA function. In some embodiments, asdRNA is designed for targeting one of the aforementioned small non-coding RNAs. In some embodiments, asdRNA is designed for targeting miRNA. In some embodiments, asdRNA is designed for targeting pre-miRNA. In some embodiments, asdRNA is designed for targeting pri-miRNA. In some embodiments, asdRNA is designed for targeting lncRNA. In some embodiments, asdRNA is designed for targeting spliceosomes.

[0084] The target RNA in the cell nucleus refers to an RNA molecule that is synthesized and / or functions in the cell nucleus of the cell. According to a preferred embodiment, the target RNA in the cell nucleus of the present invention includes lncRNA, non-coding RNA, pre-mRNA and pre-miRNA. The term "pre-mRNA" used herein refers to an unprocessed or partially processed mRNA precursor, containing introns and exons, which is synthesized by transcription from a cellular DNA template. Pre-mRNA needs to splice (remove) introns to produce an mRNA molecule containing only exons. In some embodiments, asdRNA is designed to target pre-mRNA. The terms "mitochondrial messenger RNA" and "mt-mRNA" refer to mRNA molecules transcribed from mitochondrial DNA. In some embodiments, asdRNA is designed to target mt-mRNA in mitochondria.

[0085] As used herein, the terms "isolated" or "purified" refer to material that is substantially free from components that normally accompany it 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] As used herein, the term "interval" refers to a segment having different types of adjacent segments, e.g., different types of nucleotides or nucleotide analogs, or different modifications of the same type of nucleotides or nucleotide analogs. In various embodiments of the present invention, a "deoxynucleotide monomer spacer (ISD)" refers to a segment of deoxyribonucleotides in an oligonucleotide chain having one or more deoxynucleotides and connected to at least one segment of a different type than the deoxynucleotide. For example, if the deoxynucleotide is unmodified, the different type of segment can be a ribonucleotide or an analog thereof, a modified ribonucleotide, a modified deoxynucleotide, or a deoxynucleotide analog. If the deoxynucleotide is modified, the different type of segment can be a ribonucleotide or an analog thereof, a modified ribonucleotide, an unmodified deoxynucleotide, a differently modified deoxynucleotide, or a different type of deoxynucleotide analog.

[0087] As used herein, "regulate," "modulate," and their grammatical equivalents refer to an increase or decrease (e.g., silence), in other words, upregulate or downregulate. As used herein, "gene silencing" refers to a decrease in gene expression, and can refer to a decrease in gene expression of a target gene by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0088] As used herein, the terms "inhibit," "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 the target function (e.g., production of a protein, or phosphorylation of a molecule). In certain embodiments, inhibition may refer to a reduction in target activity by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. When used in the context of a disorder or disease, the term refers to successfully preventing the onset of symptoms, alleviating symptoms, or eliminating the disease, condition (disease), or disorder.

[0089] As used herein, the term "substantially complementary" or "complementary" refers to complementarity in the double-stranded region of base pairing between the two chains with connected nucleosides, rather than any single-stranded region (e.g., overhangs at the ends or gaps between the two double-stranded regions). Complementarity need not be complete; for example, there may be any number of base pair mismatches between the two chains with connected nucleosides. However, if the number of mismatches is so large that hybridization does not occur even under the least stringent hybridization conditions, the sequence is not a substantially complementary sequence. Specifically, when two sequences are referred to as "substantially complementary" in this article, it is meant that these sequences are sufficiently complementary to each other to hybridize under the selected reaction conditions. The relationship between nucleic acid complementarity and hybridization stringency sufficient to achieve specificity is well known in the art. Two substantially complementary chains can be, for example, fully complementary, or can contain one to multiple mismatches, as long as the hybridization conditions are sufficient to allow, for example, to distinguish between paired and unpaired 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%, or any number therebetween, of base pair complementarity in the double-stranded region.

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

[0091] As used herein, the term "targeting region" refers to a region in an oligonucleotide chain that is substantially or completely complementary to another oligonucleotide chain, such that under appropriate conditions, the two chains hybridize or anneal to each other at the targeting region. For example, the antisense strand can include a targeting region through which it can hybridize to a target mRNA.

[0092] The terms "administer," "administering," and "administration" are used herein in their broadest sense. These terms refer to any method of introducing a compound or pharmaceutical composition described herein to a subject, and can include, for example, introducing the compound to a subject systemically, topically, or in situ. Thus, the production of a compound disclosed herein from a composition (whether or not the composition includes the compound) in a subject is encompassed by these terms. When these terms are used in conjunction with "systemic" or "systemically," they generally refer to systemic absorption or accumulation of a compound or composition in the blood, followed by distribution throughout the body.

[0093] As used herein, the terms "effective amount" and "therapeutically effective amount" refer to an amount of a compound or pharmaceutical composition described herein that is sufficient to affect a desired outcome, including, but not limited to, disease treatment, as shown below. In some embodiments, a "therapeutically effective amount" refers to an amount that is effective for: detectably killing or inhibiting the growth or spread of cancer cells, the size or number of tumors, 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 that is administered systemically, topically, or in situ (e.g., the amount of a compound produced in situ in a subject). A therapeutically effective amount can vary depending on the intended application (in vitro or in vivo) or the subject and disease condition being treated (e.g., the weight and age of the subject, the severity of the disease condition, the mode of administration, etc.), which can be readily determined by one of ordinary skill in the art. The term also applies to doses that induce a specific response in target cells, for example, reducing cell migration. The specific dosage may vary depending on, for example, the particular pharmaceutical composition, the subject and their age and existing health condition or risk of health condition, the dosage regimen to be followed, the severity of the disease, whether it is administered in combination with other agents, the time of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

[0094] The term "cancer" refers to the presence of cells in a subject that have typical characteristics of a cancerous cell, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain morphological characteristics. Typically, cancer cells will be present in the form of a tumor or mass, but cancer cells may also be present alone in a subject or may 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 and neck cancer, cutaneous melanoma or intraocular melanoma, breast cancer, uterine cancer, ovarian cancer, peritoneal cancer, colon cancer, rectal cancer, colorectal adenocarcinoma, cancer of the anal region, stomach cancer, gastric cancer, gastrointestinal cancer, gastric adenocarcinoma, adrenocortical cancer, and ovarian cancer.

[00135] The term "cancer" includes, but is not limited to, ovarian cancer ... For example, the general term urological cancer includes bladder cancer, prostate cancer, kidney cancer, testicular cancer, etc., while another general term, hepatobiliary cancer, includes liver cancer (which itself is a general term that includes hepatocellular carcinoma or bile duct cancer), gallbladder cancer, bile duct cancer, or pancreatic cancer. The present disclosure encompasses urological cancer and hepatobiliary cancer and includes them in the term "cancer."

[0095] The term "pharmaceutical composition" refers to a formulation containing a molecule or composition as disclosed herein as an active ingredient, typically mixed with other substances (e.g., a pharmaceutical carrier, such as sterile water) to form a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk form or in unit dosage form. A unit dosage form is any of a variety of forms, including, for example, capsules, IV bags, tablets, single pumps on aerosol inhalers, or vials. The amount of active ingredient in a unit dose of the composition is an effective amount and varies depending on the specific treatment involved. Those skilled in the art will appreciate that it is sometimes necessary to make routine adjustments to the dosage based on the patient's age and condition. The dosage will also depend on the route of administration. Various routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, etc. Dosage forms for topical or transdermal administration of the asdRNA molecules of the present invention include powders, sprays, ointments, pastes, creams, emulsions, gels, solutions, patches, and inhalants.

[0096] The term "pharmaceutical agent" refers to 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 a compound. Certain such carriers enable pharmaceutical compositions to be formulated into, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and lozenges for oral ingestion by a subject. Certain such carriers enable pharmaceutical compositions to be formulated for injection, infusion, or topical administration. For example, a pharmaceutically acceptable carrier can be a sterile aqueous solution.

[0098] The term "pharmaceutically acceptable derivative" includes derivatives of the compounds described herein, such as solvates, hydrates, esters, prodrugs, polymorphs, isomers, isotopically labeled variations, 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 unwanted 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 include, but are not limited to, acid addition salts including hydrochlorides, hydrobromides, phosphates, sulfates, bisulfates, alkylsulfonates, arylsulfonates, acetates, benzoates, citrates, maleates, fumarates, succinates, lactates, and tartrates; alkali metal cation salts such as Na, K, Li, alkaline earth metal salts such as Mg or Ca, or organic amine salts. In particular, sodium salts of oligonucleotides have been shown to be useful and are generally accepted for therapeutic administration to humans. Therefore, in one embodiment, the compounds described herein are in the form of sodium salts.

[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., that is the recipient of a particular treatment. Generally, with respect to human subjects, the terms "subject" and "patient" are used interchangeably herein.

[0101] As used herein, terms such as "treating," "treatment," "to treat," "alleviating," or "to alleviate" refer to (1) therapeutic measures that cure, slow, alleviate symptoms, and / or halt the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent or slow the progression of a targeted pathological condition or disorder. Those in need of treatment therefore include those already suffering from the disorder; those susceptible to developing the disorder; and those in need of prevention of the disorder. A subject is successfully "treated" according to the methods of the present invention if the subject exhibits one or more of the following: a decrease in the number of cancer cells or the complete absence of cancer cells; a decrease in tumor size; an inhibition or absence of cancer cell infiltration into peripheral organs (including spread of cancer into soft tissue and bone); an inhibition or absence of tumor metastasis; an inhibition or absence of tumor growth; amelioration of one or more symptoms associated with a particular cancer; a reduction in morbidity and mortality; and an improvement in quality of life.

[0102] As used herein, the term "carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, e.g., a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, which participates in or is capable of carrying or transporting the subject drug 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 excipients, 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, ethylcellulose 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 glycerol, 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; ethanol; phosphate buffered saline; and other nontoxic, compatible substances used 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 compositions. 2. Certain Implementation Options

[0103] Certain embodiments of the present invention provide a duplex RNA composition in which both the antisense strand and the sense strand are composed of linked nucleoside monomers. At least 50% of the nucleoside monomers in the entire double-stranded molecule are ribonucleoside monomers, some of which are ribonucleoside monomers and / or internucleoside bonds that are contained therein may be modified, i.e., modifications of structures derived from natural RNA. The duplex RNA of the present invention further comprises one or more deoxynucleoside monomers in an interdeoxynucleoside monomer segment (ISD). One or more ISDs may be present in the antisense strand or the sense strand, or in both. In some embodiments, each ISD is independently composed of one deoxynucleoside monomer, or is composed of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive deoxynucleoside monomers. In some embodiments, an ISD has at least two consecutive and linked deoxynucleoside monomers.

[0104] The antisense strand and the sense strand of the duplex molecule of the present invention are both relatively short, wherein the antisense strand is relatively longer than the two, and thus is referred to as "asymmetric short duplex RNA (asdRNA)".

[0105] The exemplary structures and sequences of the duplex molecules of the present invention are shown in the figures. Figure 2A In all duplex molecules, the ISD is present in the longer antisense strand.

[0106] In some embodiments, the asymmetry in length between the antisense and sense strands results in a 5' end of the antisense strand (e.g., Figure 2A ) or its 3' end (e.g., Figure 2A In other embodiments, both ends of the antisense strand have overhangs (e.g., Figure 2A (the last 13 structures on the right side of the figure).

[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 single asdRNA molecule with a cell or administering it to a subject; (ii) contacting different types of asdRNA molecules with a cell at different times or administering them to a subject at different times; or (iii) contacting different types of asdRNA molecules with a cell or administering them to a subject simultaneously.

[0108] In certain embodiments, the antisense strand comprises a region of nucleobase sequence, referred to as a "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 a target segment of a targeted target gene, wherein the target gene includes mRNA and non-coding RNA. In certain embodiments, the antisense strand comprises a nucleobase sequence that is fully complementary to a target segment of a targeted target gene. In certain embodiments, the antisense strand comprises a nucleobase sequence that, when hybridized with a target segment of a targeted target gene, contains no more than 1, 2, or 3 mismatches. In certain embodiments, the target gene is selected from an mRNA or non-coding RNA associated with a mammalian disease. In certain embodiments, at least one ISD is located within the targeting region of the antisense strand. In certain embodiments, at least one ISD is located more centrally on the antisense strand (ie, at least 1, 2, 3, 4, or 5 nucleobases from either end of the strand, ie, at position 2 or more centrally from the end of the strand).

[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 nucleomonomers, or the equivalent thereof, or a range of lengths encompassed by any two of the foregoing values ​​(both endpoints of the range are included). For example, some ranges of first strand (antisense strand) lengths include: 8-50 nucleotide monomers; 8-36 nucleotide monomers; 8-33 nucleotide monomers; 10-30 nucleotide monomers; 10-29 nucleotide monomers; 12-29 nucleotide monomers; 12-28 nucleotide monomers; 12-26 nucleotide monomers; 12-25 nucleotide monomers; 13-25 nucleotide monomers; 13-24 nucleotide monomers; 13-23 nucleotide monomers; 15-23 nucleotide monomers; 10-36 nucleotide monomers; 12-36 nucleotide monomers; 12-32 nucleotide monomers; 14-36 nucleotide monomers; and at least 8 nucleotide monomers.

[0110] In certain embodiments, the antisense strand is 10 to 36 (both ranges inclusive) nucleomonomers in length. In other words, the antisense strand is 10 to 36 (both ranges inclusive) linked nucleobase monomers in length. 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 (both ranges inclusive) linked nucleobases.

[0111] In certain embodiments, the antisense strand consists of 13-23 (both end points of the range are included) linked nucleomonomers. In certain embodiments, the antisense strand consists of 23 linked nucleomonomers. In certain embodiments, the antisense strand consists of 20 linked nucleomonomers. In certain embodiments, the antisense strand consists of 16 linked nucleomonomers.

[0112] In certain embodiments, the sense strand comprises a nucleobase sequence that is substantially complementary to the antisense strand, and its nucleobase sequence is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary (based on the total nucleobase sequence of the sense strand) to the sequence of the region to which the antisense oligonucleotide is attached. These substantially complementary sequences from the two strands form one or more double-stranded regions. In certain embodiments, the sense strand has a nucleobase sequence that is fully complementary to the sequence of the region to which the antisense strand is attached. In certain embodiments, at least one ISD can be located anywhere in the sense strand. In certain embodiments, the ISD is located in the double-stranded region of the sense strand. In some embodiments, the ISD is located in the more central portion of the sense strand (i.e., at least 1, 2, 3, 4, or 5 nucleobases from either end of the strand, i.e., at position 2 or more central from the end of the strand). In certain embodiments, the ISD is not required to be distributed in the sense strand.

[0113] In one feature, the sense strand is shorter than the antisense strand, provided that the sense strand is capable of forming a thermodynamically stable duplex with the antisense strand. In certain embodiments, the sense strand is about one-half to one nucleotide shorter than the antisense strand. In certain embodiments, the sense strand is about one-quarter to one nucleotide shorter than the antisense strand. In certain embodiments, the sense strand is 6 to 35 (both ranges inclusive) nucleotide monomers in length. In other words, the sense strands are 6 to 35 (both ranges inclusive) linked nucleobase monomers. 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 (both ranges inclusive) linked nucleobase monomers. In certain embodiments, the sense strand comprises an oligonucleotide consisting of linked nucleobases of length 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, or a range consisting of any two of the above values. In some embodiments, the sense strand is a sense oligonucleotide.

[0114] In one feature, the sense strand has a backbone length that is 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 nucleotides shorter than the antisense strand. 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 the equivalent thereof, or a range of lengths encompassed by any two of the foregoing values ​​(both endpoints of the range are inclusive). For example, in certain embodiments, some ranges of the sense strand include: 6-49 nucleotide monomers; 8-46 nucleotide monomers; 8-35 nucleotide monomers; 9-35 nucleotide monomers; 10-46 nucleotide monomers; 10-40 nucleotide monomers; 10-34 nucleotide monomers; 8-32 nucleotide monomers; 8-30 nucleotide monomers; 8-29 nucleotide monomers; 9-29 nucleotide monomers; 9-26 nucleotide monomers; 9-25 nucleotide monomers; 10-29 nucleotide monomers; 10-28 nucleotide monomers; 10-26 nucleotide monomers; 10-25 nucleotide monomers; 11-24 nucleotide monomers; 11-23 nucleotide monomers; 12-23 nucleotide monomers; 13-23 nucleotide monomers; 12-22 nucleotide monomers; 13-23 nucleotide monomers; 15-23 nucleotide monomers and at least 6 nucleotide monomers. In certain embodiments, the second strand can have a backbone length of any number of nucleotide monomers provided that the second strand is capable of forming a thermodynamically stable duplex with the first strand.

[0115] In certain embodiments, the sense strand is shorter than the antisense strand by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleomonomers. In certain embodiments, the sense strand consists of 8-23 linked nucleomonomers (both ends of the range are inclusive). In certain embodiments, the sense strand consists of 13 linked nucleomonomers. In certain embodiments, the sense strand consists of 14 linked nucleomonomers.

[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 5' blunt end, a 5' overhang and a 3' blunt 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 1-15, 1-10, 1-9, 1-8, or 1-5 nucleotide monomers (both ranges are inclusive).

[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 1-15, 1-10, 1-9, 1-8, or 1-5 nucleotide monomers (both ranges are inclusive).

[0119] In one embodiment of the invention, the antisense strand has a 3' overhang of 1-15 (both end values ​​of the range are included) nucleotide monomers and a 5' overhang of 1-15 (both end values ​​of the range are included) nucleotide monomers. In another embodiment, the antisense strand has a 3' overhang of 1-26 (both end values ​​of the range are included) nucleotide monomers and a 5' blunt end or a 5' recessed end. In another embodiment, the antisense strand has a 5' overhang of 1-26 (both end values ​​of the range are included) nucleotide monomers and a 3' blunt end or a 3' recessed end.

[0120] In various embodiments of the present invention, the two ends of the second strand (sense strand) are 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 1-15, 1-10, 1-9, 1-8, or 1-5 (both end points of the range are included) nucleotide monomers. 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 1-15, 1-10, 1-9, 1-8, or 1-5 nucleotide monomers.

[0121] In the asdRNA molecules of the present invention, at least one nucleotide monomer in the first and / or second strands can be a modified nucleotide or nucleotide analog, such as a sugar-modified, backbone-modified, and / or base-modified nucleotide. In one embodiment, the backbone-modified nucleotide has at least one modification in the internucleoside linkage, such as including at least one of a nitrogen heteroatom or a sulfur heteroatom. In some embodiments, the modified internucleoside linkage is or comprises: a phosphorothioate group (P=S), a phosphotriester, a methylphosphonate, or a phosphoramidate.

[0122] In certain embodiments, antisense strand and / or sense strand comprise at least one modified internucleoside bond.This modified internucleoside bond can be between two ribonucleoside monomers, two deoxyribonucleoside monomers or a deoxyribonucleoside monomer and a ribonucleoside monomer.Alternatively, the phosphate group on the nucleoside monomer of at least one end can be modified.In certain embodiments, internucleoside bond is a bond between thiophosphate nucleoside.In certain embodiments, internucleoside bond is a bond between thiophosphoramidate nucleoside.In certain embodiments, internucleoside bond is a bond between thiophosphate nucleoside.In certain embodiments, internucleoside bond is a bond between each nucleoside of oligonucleotide chain.In certain embodiments, all internucleoside bonds in chain (antisense strand or sense strand or both) are bonds between thiophosphate nucleoside, or the mixing of thiophosphate bond and phosphodiester bond.

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

[0124] In certain embodiments, the 2' position of the modified sugar moiety is substituted with a group selected from the group consisting of OR, R, halogen, SH, SR, NH2, NHR, NR2, or CN, wherein each R is independently C1-C6 alkyl, alkenyl, or alkynyl, and halogen is F, Cl, Br, or I. In some embodiments, the 2' position of the modified sugar moiety is substituted with a group selected from the group consisting of allyl, amino, azido, thio, O-allyl, O-C1-C6 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 ), where each R1, R m and R n are independently H, or substituted or unsubstituted C1-C 10 In some embodiments, the modified sugar moiety has a substituent group selected from the group consisting of 5'-vinyl, 5'methyl (R or S), 4'-S, 2'-F, 2'-OCH3, 2'-OCH2CH3, 2'-OCH2CH2F, and 2'-O(CH2)2OCH3. In some embodiments, the modified sugar moiety is substituted with a bicyclic sugar selected from the group consisting 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(CH2OCH3)—O-2′, 4′-C(CH3)(CH3)—O-2′, 4′-CH2—N(OCH3)-2′, 4′-CH2—O—N(CH3)-2′, 4′-CH2—N(R)—O-2′ (wherein R is H, C1-C 12 alkyl or 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 consisting of a 2'-O-methoxyethyl modified sugar (MOE), a 4'-(CH2)—O-2' bicyclic sugar (LNA), a 2'-deoxy-2'-fluoroarabinose (FANA), and a methyl(methyleneoxy)(4'-CH(CH3)—O-2) bicyclic sugar (cEt).

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

[0127] In some embodiments, the modified nucleobase is selected from the group consisting of 5-methylcytosine (5-Me-C), hypoxanthine nucleobases, 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-methylpseudouracil, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3) uracil and cytosine and other pyrimidine bases. Alkynyl derivatives, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo (especially 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0128] In certain embodiments, the modified nucleobase in the molecules of the invention is 5-methylcytosine. In one embodiment, each cytosine base in the molecules of the 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, the antisense strand, the sense strand, or both strands of the molecules of the present invention comprise linked ribonucleoside monomers. In certain embodiments, the entire antisense strand or the entire sense strand consists solely of linked ribonucleoside monomers. In certain embodiments, the entire sense strand consists solely of linked ribonucleoside monomers. In one feature, the antisense strand, the sense strand, or both strands, in addition to the linked ribonucleoside monomers, further comprise an ISD comprised of one or more linked deoxyribonucleoside monomers. In certain embodiments, the antisense strand, the sense strand, or both strands, in addition to the linked ribonucleoside monomers, further comprise an ISD comprised 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 certain features, there may be more ISD segments. The ISD may be located at any position in either strand. In certain embodiments, one or more ISDs are inserted into a ribonucleoside monomer fragment to separate the ribonucleoside monomer fragment into multiple fragments. In certain embodiments, each ISD is independently composed of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 linked deoxynucleotide monomers.

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

[0131] In certain embodiments, the total number of deoxynucleotide monomers in an asdRNA molecule does not exceed the total number of ribonucleotide monomers in the same asdRNA molecule. In certain embodiments, the total number of deoxynucleotide monomers in any one strand of an asdRNA molecule does not exceed the total number of ribonucleotide monomers in the same strand. In certain embodiments, the total number of deoxynucleotide monomers in a first strand of an asdRNA molecule does not exceed the total number of ribonucleotide monomers in the same first strand. The total number of deoxynucleotide monomers in a second strand of an asdRNA molecule does not exceed the total number of ribonucleotide monomers in the same second strand. In certain embodiments, the total number of deoxynucleotide monomers in an asdRNA molecule does not exceed the total number of ribonucleotide monomers in the same asdRNA molecule, and the total number of deoxynucleotide 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 linked deoxynucleotide monomer of the ISD is a modified deoxynucleotide or deoxynucleotide analog. Deoxynucleotides can be modified in the same or similar manner to have modified internucleoside linkages, modified sugar moieties, and / or modified nucleobases.

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

[0134] In some embodiments, the sugar portion 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 in each ISD in the antisense strand, the sense strand, or both strands has a sugar moiety modified with 2'-deoxy-2'-fluoroarabinose (FANA). In another embodiment, all nucleoside monomers in the ISD have a sugar moiety modified with FANA. In another embodiment, all nucleosides in the ISD are naturally occurring deoxyribonucleosides. In one embodiment, all nucleosides in the ISD are naturally occurring deoxyribonucleosides or have a sugar moiety modified with 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, wherein the modified sugar moiety is selected from the group consisting of a 2'-O-methoxyethyl modified sugar (MOE), a 4'-(CH2)—O-2' bicyclic sugar (LNA), and a methyl(methyleneoxy)(4'-CH(CH3)—O-2) bicyclic sugar (cEt).

[0136] In certain embodiments, each internucleoside linkage between the deoxyribonucleoside monomers of each ISD is a phosphorothioate linkage. In certain embodiments, each internucleoside linkage between the deoxyribonucleoside monomers of each ISD is a native phosphate linkage that is not modified by phosphorothioate.

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

[0138] In certain embodiments, the molecules of the invention have an antisense strand or a sense strand composed of ribonucleoside monomers, wherein each internucleoside bond is a phosphorothioate bond. In certain embodiments, the molecules of the invention have an antisense strand or a sense strand composed of ribonucleoside monomers, wherein each nucleoside bond is a native phosphate bond that is not modified by phosphorothioate.

[0139] In certain embodiments, molecules of the invention comprise a sense strand, wherein each nucleomonomer of the sense strand comprises the same modification as the complementary nucleomonomer of the antisense strand.

[0140] An exemplary structure of a molecule of the present invention having an antisense oligonucleotide strand and a sense oligonucleotide strand is shown in FIG. Figure 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 can achieve powerful gene silencing. The data shown in all of the following examples indicate that the new platform technology based on the present invention, i.e., asymmetric duplexes of antisense oligoribonucleotides with at least one ISD, can achieve extremely powerful gene silencing. Further research has been conducted on the SAR (structure-activity relationship) characteristics of asdRNA, including the length of the motif, the ISD motif, various modifications, etc., which help to identify various structural design factors that may affect gene silencing activity. These SAR factors are very important for designing optimized gene silencers to target various sequences and structures of more than 100,000 different mRNAs and more non-coding RNAs in typical mammalian cells. Our data on asdRNA gene silencing activity and SAR show that the gene silencing characteristics of asdRNA are very different from those of siRNA and ASO, which suggests that a novel and unique mechanism of gene silencing mechanism has yet to be elucidated.

[0142] In certain embodiments, the molecules of the present invention can be stabilized to prevent degradation by at least one chemical modification or secondary structure. Sense oligonucleotide chains and antisense oligonucleotide chains can have unpaired or imperfectly paired nucleotide monomers. Sense oligonucleotide chains and / or antisense oligonucleotide chains can have one or more nicks (nicks in the nucleic acid backbone), gaps (fragment chains with one or more missing nucleotides) and modified nucleotides or nucleotide analogs. Not only any or all nucleotide monomers in sense oligonucleotide chains and antisense oligonucleotide chains can be chemically modified, but each chain can be conjugated to one or more parts or ligands to enhance its functionality, for example, with a part or part selected from: polypeptide, antibody, antibody fragment, polymer, polysaccharide, lipid, hydrophobic part or molecule, cationic part or molecule, lipophilic compound or part, oligonucleotide, cholesterol, GalNAc and nucleic acid aptamer.

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

[0144] In certain embodiments, the target is an mRNA, pre-mRNA, mt-mRNA, or non-coding RNA associated with a mammalian disease. In certain embodiments, the target is an mRNA. In certain embodiments, the target is a pre-mRNA. In certain embodiments, the target is a non-coding RNA, such as a microRNA and a lncRNA. In certain embodiments, the target is an mt-mRNA. As long as the antisense strand is substantially complementary to the target sequence, the antisense strand hybridizes with the target sequence to occupy the target, thereby inactivating the target gene. 3. Unpaired or mismatched regions

[0145] The complementary region between the antisense strand and the sense strand of the asdRNA of the present invention can have at least one unpaired or imperfectly paired region, for example, one or more mispairings. Mispairings in the sense strand are sometimes required to reduce off-target effects or to achieve other functions of the asdRNA.

[0146] As is well known to those skilled in the art, mispairing bases can be introduced without eliminating activity. Similarly, the antisense strand of asdRNA of the present invention can include the zone of unpairing or mispairing when with the target RNA base pairing. Sometimes need the mispairing in the antisense strand to reduce the effect of missing the target or realize other functions of asdRNA. 4. Modification

[0147] Nucleoside monomer is a kind of base-sugar composition.The core base (also referred to as base) part of nucleoside monomer is normally heterocyclic base part.Nucleoside monomer is the nucleoside monomer that further comprises the phosphate group that is covalently attached to the sugar part of nucleoside.For those nucleotide monomers that comprise pentofuranosyl sugar, the phosphate group can be attached to the 2 ', 3 ' or 5 ' hydroxyl part of sugar.Oligonucleotide is formed by adjacent nucleoside monomers being covalently attached to each other, to form linear polymeric oligonucleotide.In oligonucleotide structure, phosphate group is commonly referred to as the internucleoside bond that forms oligonucleotide.

[0148] Modification of asdRNA molecule of the present invention, antisense strand and / or sense strand comprises replacement or the change of internucleoside bond, sugar moiety or core base.Modified asdRNA, antisense strand and / or sense strand are more preferred than its native form in some cases because ideal characteristic, for example the inhibitory activity that increases, the cellular uptake that enhances, the chain affinity, the solubility that increase, reduce non-specific interactions, and the resistance to RNase degradation or the stability that enhances.Therefore, usually can obtain the result similar to the short antisense strand of the nucleoside monomer with this type of chemical modification.One or more natural nucleotides in antisense strand of the present invention and the sense strand can be replaced by modified nucleotide or nucleotide analog.Replacement can occur in any site of antisense strand and sense strand.

[0149] Modifications of oligonucleotides have been studied to improve the stability of various oligonucleotides, including antisense oligonucleotides, ribozymes, aptamers, and RNAi (Chiu and Rana, 2003; Czauderna et al., 2003; deFougerolles 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. Within oligonucleotide molecules, chemical modifications can be introduced into the phosphate backbone (e.g., phosphorothioate linkages), sugars (e.g., locked nucleic acids, glycerol nucleic acids, cEt, 2'-MOE, 2'-fluorouridine, 2'-O-methyl), and / or bases (e.g., 2'-fluoropyrimidine).

[0151] Several 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, backbone-modified, and / or base-modified nucleotide. 4.1 Modified Internucleoside Bonds or Backbone-Modified Nucleotides

[0153] The naturally occurring internucleoside linkage in RNA and DNA is a 3' to 5' phosphodiester bond. AsdRNA molecules of the invention having one or more modified internucleoside linkages (i.e., non-naturally occurring internucleoside linkages) in one or both chains are sometimes selected for desirable properties (e.g., enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases) compared to corresponding molecules having only naturally occurring internucleoside linkages.

[0154] The oligonucleotide chain of key between modified nucleosides comprises the key between the nucleosides that retains phosphorus atom and the key between the nucleosides that does not have phosphorus atom.In one embodiment, the key between phosphodiester nucleosides can be modified to comprise at least one in nitrogen heteroatom or sulfur heteroatom.Representational phosphorus-containing nucleosides key includes, but is not limited to: phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, thiophosphoramidate and thiophosphate.The method for preparing phosphorus-containing and non-phosphorus-containing key is well-known.

[0155] In one embodiment, the modified nucleotide or nucleotide analog is a modified nucleotide of the backbone. The modified nucleotide of the backbone may have a modification on the phosphodiester internucleoside bond. In another embodiment, the modified nucleotide of the backbone is a thiophosphate internucleoside bond. In certain embodiments, each internucleoside bond is a thiophosphate internucleoside bond. 4.2 Modified sugar moieties

[0156] The antisense strand and / or sense strand of the present invention may optionally contain one or more nucleoside monomers with modified sugar moieties. These sugar-modified nucleoside monomers can impart enhanced nuclease stability, increased binding affinity, or some other favorable biological properties to the antisense strand and / or sense strand. In certain embodiments, the nucleoside monomer comprises a chemically modified ribofuranose ring portion. Examples of chemically modified ribofuranose rings include, but are not limited to: the addition of substituents; including 5' and 2' substituents, non-geminal ring atoms bridged to form bicyclic nucleic acids (BNAs), replacement of ribose ring oxygen atoms with S, N(R) or C(R1)(R2) (R, R1 and R2 are each independently H, C1-C12 alkyl or protecting groups), and combinations thereof. Examples of chemically modified sugars include 2'-F-5'-methyl substituted nucleosides (see PCT International Application No. WO 2008 / 101157 published on August 21, 2008 for other disclosed 5',2'-disubstituted nucleosides), or replacement of the ribosyl ring oxygen atom with S and further substitution at the 2'-position (see, U.S. Patent Application No. US2005-0130923 published on June 16, 2005), or optional 5'-substitution for BNA (see PCT International Application No. WO 2007 / 134181 published on November 22, 2007, in which LNA is substituted with, for example, 5'-methyl or 5'-vinyl).

[0157] Examples of nucleoside monomers having modified sugar moieties include, without limitation, nucleosides comprising 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, 2'-OCH2CH3, 2'-OCH2CH2F, and 2'-O(CH2)2OCH3 substituents. The substituent at the 2' position may also be selected from allyl, amino, azido, thio, O-allyl, O-C1-C10 alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-ON(Rm)(Rn), O-CH2-C(=O)-N(Rm)(Rn), and O-CH2-C(=O)-N(R1)-(CH2)2-N(Rm)(Rn), wherein each R1, Rm, and Rn is independently H, or substituted or unsubstituted C1-C10 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 bridging group between the 4' and 2' ribosyl ring atoms. In certain embodiments, the asdRNA, antisense strand, and / or sense strand provided herein comprise one or more BNA nucleosides, the bridging group in the BNA nucleoside comprising one of the following formulae: 4'-(CH2)—O-2'(LNA), 4'-(CH2)—S-2, 4'-(CH2)2—O-2'(ENA), 4'-CH(CH3)—O-2', and 4'-CH(CH2OCH3)—O-2' (and analogs thereof, see U.S. Patent No. 7,399,845, issued July 15, 2008); 4'-C(CH3)(CH3)—O-2'(and and analogs thereof, see PCT / US2008 / 068922, published as WO / 2009 / 006478 on January 8, 2009); 4′-CH2—N(OCH3)-2′ (and analogs thereof, see PCT / US2008 / 064591, published as WO / 2008 / 150729 on December 11, 2008); 4′-CH2—O—N(CH3)-2′ (see U.S. patent application US2004-0171570, published on September 2, 2004); 4′-CH2—N(R)—O-2′, wherein R is H, C1-C 12 alkyl or protecting groups (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 the like, see PCT / US2008 / 066154, published Dec. 8, 2008, and disclosed as W2008 / 154401).

[0159] In certain embodiments, bicyclic nucleosides include, but are not limited to: (A) α-L-methyleneoxy (4′-CH2—O-2) BNA, (B) β-D-methyleneoxy (4′-CH2—O-2) BNA, (C) vinyloxy (4′-(CH2)2—O-2′) BNA, (D) aminooxy (4′-CH2—O—N(R)-2′) BNA, (E) oxyamino (4′-CH2—N(R)—O-2) BNA, (F) methyl (methyleneoxy) (4′-CH(CH3)—O-2) BNA (also known as constrained ethyl) ethyl) or cEt), (G) methylenethio (4′-CH2—S-2′) BNA, (H) methyleneamino (4′-CH2—N(R)-2′) BNA, (I) methyl carbocycle (4′-CH2—CH(CH3)-2) BNA, (J) propylene carbocycle (4′-(CH2)3-2′) BNA and (K) vinyl BNA.

[0160] In certain embodiments, the modified nucleotide or nucleotide analog is a sugar-modified ribonucleotide in which the 2'-OH group is substituted with a group selected from the group consisting of H, OR, R, halogen, SH, SR, NH2, NHR, NR2, and CN, wherein each R is independently selected from the group consisting of C1-C6 alkyl, alkenyl, or alkynyl, and a halogen selected from the group consisting of F, Cl, Br, or I. In certain embodiments, the sugar-modified ribonucleotide is selected from the group consisting 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 one embodiment, the sugar-modified deoxynucleotide is a FANA-modified deoxynucleotide.

[0161] Chemical modifications at the 2' position of the ribose sugar can stabilize the molecules of the present invention. For example, 2'-O-methylpurines and 2'-fluoropyrimidines can increase their resistance to endonuclease activity in serum. The site of introduction of the modification should be carefully selected to avoid significantly reducing the silencing / regulatory capacity of the molecule. In certain embodiments, the first nucleomonomer adjacent to the 5'-terminal nucleomonomer of the antisense strand is a 2'-fluoro ribonucleotide. 4.3 Modified Nucleobases

[0162] The antisense strand and / or sense strand in the asdRNA molecule can also have a modified or substituted nucleobase (or base). Although the nucleobase (or base) modification or replacement is structurally different from naturally occurring or synthetic unmodified nucleobases, it is functionally interchangeable therewith. Both natural and modified nucleobases can participate in hydrogen bonding. The nucleobase modification can impart nuclease stability, binding affinity or some other favorable biological properties to the asdRNA molecule. Modified nucleobases include synthetic and natural nucleobases, such as, for example, 5-methylcytosine (5-Me-C). Certain nucleobase replacements, including 5-methylcytosine replacements, are particularly useful for improving the binding affinity of antisense strand and sense strand. 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] Other modified nucleobases include, but are not limited to: 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 Pyrimidine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo (especially 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

[0164] Heterocyclic base moieties can include those in which a purine or pyrimidine base is substituted with another heterocycle, such as 7-deazaadenine, 7-deazaguanine, 2-aminopyridine, and 2-pyridone. Nucleobases particularly useful for increasing the binding affinity of the antisense and sense strands include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.

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

[0166] Any modified nucleotide or analog that may be advantageous for stability or affinity may be prepared without departing from the spirit and scope of the present invention. Several examples of such chemical modifications are the same as summarized above. 5. Pharmaceutical Compositions

[0167] In some embodiments, the present invention also provides a pharmaceutical formulation comprising an 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, etc. that are compatible with pharmaceutical administration. Suitable carriers are described in "Remington: The Science and Practice of Pharmacy, Twentieth Edition," Lippincott Williams & Wilkins, Philadelphia, PA," which is 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 carriers, such as fixed oils, may also be used. It is well known in the art that such media and agents can be used in pharmaceutically active substances. Unless any conventional media or agents are incompatible with the asdRNA molecules, they are considered for use in the composition.

[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, polysaccharide polymers, nanoparticles, nanoemulsions, cholesterol, lipids, lipophilic compounds or moieties, and lipids.

[0169] In certain embodiments, the present invention provides a method of treating a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition. In one embodiment, the pharmaceutical composition is administered by a route selected from the group consisting of intravenous injection (iv), subcutaneous injection (sc), oral administration (po), intramuscular (im), oral administration, inhalation, topical, intrathecal, and other site administration. In another embodiment, 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] Formulation methods are disclosed in PCT International Application PCT / US02 / 24262 (WO03 / 011224), US Patent Application Publication No. 2003 / 0091639, and US Patent Application Publication No. 2004 / 0071775, each of which is incorporated herein by reference.

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

[0172] These steps may involve appropriately mixing, granulating and compressing or dissolving the ingredients to obtain the desired formulation. In another embodiment, the therapeutically effective amount of the asdRNA molecule is administered in a suitable dosage form without a standard pharmaceutical carrier or diluent. In some embodiments, the therapeutically effective amount of the 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, gum arabic, magnesium stearate, stearic acid, etc. Exemplary liquid carriers include syrup, peanut oil, olive oil, water, etc. Similarly, carriers or diluents may include time delay materials known in the art, such as glyceryl monostearate or glyceryl distearate, used alone or in combination with wax, ethylcellulose, hydroxypropyl methylcellulose, methyl methacrylate, etc. Other fillers, excipients, flavorings, and other additives as known in the art may also be included in pharmaceutical compositions according to the present invention.

[0173] Pharmaceutical composition of the present invention can be prepared in a well-known manner, for example, by conventional mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, embedding or lyophilizing process. One or more physiologically acceptable carriers can be used to prepare pharmaceutical composition in a conventional manner, and the carrier includes excipients and / or adjuvants that can promote sense oligonucleotide and antisense oligonucleotide to be processed into pharmaceutically acceptable preparations. Of course, suitable preparation depends on selected route of administration.

[0174] The compositions, compounds, combinations or pharmaceutical compositions of the present invention can be applied to a subject using many well-known methods currently used for chemotherapy. For example, to treat cancer, the asdRNA molecules of the present invention can be injected directly into a tumor, injected into the bloodstream or body cavity, or administered orally or through a skin patch. For the treatment of psoriatic conditions, systemic administration (e.g., oral administration) or topical administration to the affected skin area is a preferred route of administration. The dosage selected should be sufficient to constitute an effective treatment, but not so high as to cause unacceptable side effects. During and after treatment, the disease condition (e.g., cancer, psoriasis, etc.) and the patient's health should be closely monitored within a reasonable period of time. 6. Use 6.1 Usage

[0175] The present invention also provides a method for regulating gene expression or function in a cell or organism. The cell can be a eukaryotic cell, such as a mammalian cell. The method comprises the steps of contacting the cell or organism with an asdRNA molecule disclosed herein under conditions that allow selective gene silencing to occur, and mediating selective gene silencing produced by the asdRNA molecule on a target nucleic acid having a sequence portion that is substantially complementary to the antisense strand of the asdRNA molecule. The target nucleic acid can be RNA, such as mRNA, pre-mRNA, mt-mRNA, or non-coding RNA, which either encodes a protein associated with a disease or regulates a portion of a biological pathway associated with the disease.

[0176] In one embodiment, the contacting step comprises introducing the asdRNA molecule into a target cell or organism in culture in which selective gene silencing can occur. In a further embodiment, the introducing step comprises mixing, transfection, lipofection, infection, electroporation or other delivery techniques. In another embodiment, the introducing step comprises administering the asdRNA molecule intravenously, subcutaneously, intrathecally, orally, by inhalation, topically or other clinically acceptable administration methods using a pharmaceutically acceptable excipient, carrier or diluent, wherein the pharmaceutically acceptable excipient, carrier or diluent is selected from a pharmaceutical carrier, a positively charged carrier, a liposome, a lipid nanoparticle, a protein carrier, a polymer, a nanoparticle, a nanoemulsion, a lipid, N-acetylgalactosamine (GalNAc), a lipophilic compound or moiety, and a lipid.

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

[0178] In one embodiment, the gene or RNA targeted by the compositions of the present invention is associated with a disease (e.g., a human disease or an animal disease), a pathological condition, or an adverse condition. In another embodiment, the target gene or target RNA is a gene or RNA of a pathogenic microorganism. In a further embodiment, the target gene or target RNA is a gene or RNA of viral origin. In another embodiment, the target gene or target RNA is a tumor-associated gene or RNA.

[0179] In an optional embodiment, the gene or RNA targeted by the composition of the present invention is a gene or RNA associated with the following diseases: cancer, autoimmune diseases, inflammatory diseases, degenerative diseases, infectious diseases, proliferative diseases, metabolic diseases, immune-mediated disorders, allergic diseases, skin diseases, malignancies, gastrointestinal diseases, liver diseases, respiratory disorders, cardiovascular disorders, skin diseases, kidney diseases, rheumatoid diseases, nervous system disorders, mental disorders, endocrine disorders, or diseases or disorders associated with aging. 6.2 Treatment

[0180] The present invention also provides methods for treating or preventing various diseases or conditions, wherein the various diseases or conditions include those that can be treated or prevented by ASO and siRNA (Czech, 2006; deFougerolles 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 comprises administering an effective amount of an asdRNA molecule to a subject in need thereof under conditions in which the desired gene inhibition (described in Section 6.1 above) occurs.

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

[0182] In some embodiments, the present invention can be used to treat or prevent cancer. AsdRNA compositions can be used to silence or knock down genes associated with cell proliferation disorders or malignancies. Examples of these genes are k-Ras, β-catenin, and Stat3. These oncogenes are active in and associated with a wide range of human cancers.

[0183] The novel compositions of the present invention can also be used to treat or prevent ocular 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 diseases; and inflammatory conditions (e.g., inflammatory bowel disease (IBD), arthritis, rheumatoid disease, autoimmune diseases), and skin diseases.

[0184] In another embodiment, the method of administration is selected from the group consisting of intravenous injection (iv), subcutaneous injection (sc), oral (po), intrathecal, inhalation, topical and regional administration. Example

[0185] The following examples are provided to further illustrate various features of the present invention. The examples also illustrate useful methods for practicing the present invention. These examples do not limit the claimed invention. Methods and Materials Cell culture

[0186] DLD1 cells were purchased from ATCC. DLD1 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 known to those skilled in the art can be purchased and used. asdRNA transfection cells

[0187] 24 hours before transfection, DLD1 cells, HepaRG cells or other commonly used cell lines were seeded into 6-well plates (1x10 5 cells / 2 mL / well). As described in the preparation method, AsdRNA was transfected with RNAiMAX (Thermo Fisher, USA) at a final concentration of 100 pM, 200 pM, 1 nM, 3 nM, 5 nM, 10 nM, or 30 nM. Briefly, asdRNA and RNAiMAX were incubated in serum-free OPTI-MEM (Thermo Fisher) for 20 min and then added to cells containing culture medium. Quantitative PCR

[0188] Transfected cells were harvested 48 hours after transfection with the indicated asdRNAs. RNA was isolated using TRIZOL and qRT-PCR was performed using TaqMan One-Step RT-PCR Reagents. The CTNNB1 assay (Thermo Fisher) was used for β-catenin mRNA detection; the APOCIII assay was used for APOCIII mRNA detection; the APOB assay was used for APOB mRNA detection; and the GAPDH mRNA level was used as an internal control.

[0189] Target sequence

[0190] In order to study the gene silencing effect of the asdRNA disclosed in the present invention, asdRNAs targeting different genes were designed and produced. Figure 1 Shown are exemplary target genes, target sequences, and the corresponding antisense strands of asdRNAs that were designed and used. Example 1: Structure-activity relationship of asdRNA with ISD distributed only in AS and SS of pure RNA with different lengths and positions Relationship (SAR) research

[0191] Figure 2A The structures of a series of embodiments of asdRNAs in which the ISD is present only in the AS are shown. By keeping the antisense strand (AS) containing the ISD constant and varying the annealing position and length of the sense strand (SS) consisting only of linked ribonucleoside monomers, the ISD can be expressed in the AS. Figure 2A sdRNA-a1 to -a33 targeting the APOCIII gene were designed (their sequences are shown in FIG. Figure 2B In addition, single-stranded antisense oligonucleotides (ASOs) with the same structure and sequence as the antisense strand of the asdRNA were also designed as corresponding single-stranded ASOs for comparison. The gene silencing activity of these asdRNAs and corresponding single-stranded ASOs at 100 pM was tested in HepaRG cells.

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

[0193] exist Figure 2B In the sequence, all lowercase letters "a, c, g, t" represent DNA residues; all uppercase letters "A, C, G, U" represent RNA residues modified by 2'-MOE; all underlined uppercase letters " A 、 C 、 G 、 U ” represents RNA residues, wherein all “U” are 5-methyluridine 2'-MOE RNA residues; wherein all “ U ” are all 5-methyluridine RNA residues; all “C” are 5-methylcytosine 2'-MOE RNA residues; all “c” are 5-methylcytosine DNA residues; all “ C ” are 5-methylcytosine RNA residues; all “*” in the sequence represent PS (phosphorothioate internucleoside linkage).

[0194] Figure 2C The gene silencing results shown in indicate that all designed asdRNAs have extremely strong gene silencing activity against APOCIII at extremely low concentrations (picomolar levels) and are stronger and more effective than the corresponding single-stranded ASO (i.e., ISIS304801) optimized using state-of-the-art technology. Example 2: Structure-activity relationship (SAR) study of asdRNA with ISD in both AS and SS

[0195] Figure 3A Another series of different structural designs of asdRNAs are shown. In these asdRNAs, the SS containing the ISD remains unchanged, while () ISDs containing various numbers of deoxynucleotide monomers are distributed at different positions of the antisense strand (labeled as sdRNAb1-b4, with structures and sequences as shown in FIG. Figure 3A In addition, single-stranded antisense oligonucleotides with the same structure and sequence as the antisense strands of asdRNAb1-b4 were also designed as single-stranded ASOs corresponding to each asdRNA for comparison. The gene silencing activity of asdRNAb1-b4 targeting APOCIII and each corresponding ASO at 100 pM was tested in HepaRG cells (the comparison results are shown in FIG. Figure 3B shown).

[0196] exist Figure 3A In the diagram, all letters "D", "R" and Figure 2A Represent the same meaning, all lowercase letters "a, c, g, t", "A, C, G, U" and "*" in the illustrated sequence are the same as Figure 2B The same meaning is expressed in .

[0197] The results showed that all designed ASs containing at least one ISD had efficient gene silencing activity at extremely low concentrations (picomolar level) and were significantly stronger and more effective than the corresponding ASOs.

[0198] Example 3: SAR Study of asdRNA Containing ISD in AS and Unmodified Internucleoside Bonds

[0199] Figure 4A The exemplary structural designs of a series of embodiments of asdRNA targeting APOB gene are shown, wherein the ISD is completely distributed in AS, and each internucleoside bond between adjacent nucleoside monomers in these asdRNA molecules is a naturally occurring internucleoside bond. By keeping AS unchanged, the annealing position and length of the SS composed entirely of ribonucleoside monomers are changed (labeled as sdRNA-c1 to -c8). The gene silencing effect of these structural variants at 5nM was tested in HepaRG cells. The results are shown in FIG. Figure 4B shown.

[0200] exist Figure 4A In the diagram, all letters "D", "R" and "rR" are the same as Figure 2A The same meanings are expressed in the asdRNA sequence shown in the figure; all lowercase letters "a, c, g, t", uppercase letters "A, C, G, U", underlined uppercase letters " A 、 C 、 G 、 U ” Figure 2B The meanings expressed in have the same meaning.

[0201] Specifically, in HepaRG cells, sdRNA-c1 to -c8 (structure and sequence as shown in Figure 4A Each of the two compounds (shown in Figure 2) exhibited strong gene silencing activity against the expected target APOB gene at a concentration of 5 nM.

[0202] Example 4: Gene Silencing Efficacy of asdRNA Targeting β-Catenin

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

[0204] The results in Examples 1-4 strongly indicate that asdRNA designed according to the principles of the present invention can target different genes through different target sequence motifs, thereby achieving great gene silencing efficacy.

[0205] In other embodiments of detecting the gene silencing effect of the asdRNA disclosed in the present invention, the asdRNA was designed to target pre-mRNA in the cell nucleus, lncRNA in the cell nucleus, and mt-mRNA in the mitochondria. The detection and quantitative PCR results were performed using the same method as in the above embodiments. Both showed that the asdRNA designed according to the principles of the present invention can achieve great gene silencing efficacy, while the corresponding aiRNA / siRNA targeting the same RNA in the cell nucleus and mitochondria failed to show gene silencing activity. Equivalent

[0206] The representative examples are intended to help illustrate the present invention and are not intended to, and should not be construed to, limit the scope of the present invention. Indeed, various modifications of the present invention and numerous further embodiments thereof, in addition to those shown and described herein, will be apparent to those skilled in the art from the entire contents of this document, including the examples and the citations to the scientific and patent literature contained herein. The examples contain important additional information, illustrations, and guidance that can be applied in practicing the various embodiments of the present invention and their equivalents.

[0207] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill 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 the present disclosure, the preferred methods and materials are now described. The methods described herein can be performed in any order that is logically possible, except in the specific order disclosed. Incorporated by Reference

[0208] Reference and citation have been made to other documents in this disclosure, such as patents, patent applications, patent publications, periodicals, books, papers, and online content. For all purposes, all such documents are incorporated herein by reference in their entirety. Any material or portion thereof incorporated herein by reference that conflicts with existing definitions, statements, or other public materials explicitly set forth herein is incorporated only to the extent that the incorporated material does not conflict with the present disclosure. In the event of a conflict, the material or portion thereof supporting the present disclosure will be disclosed as preferred to resolve the conflict. References 1.Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, TuschlT.Duplexes of21-nucleotide RNAs mediate RNA interference in culturedmammalian cells.Nature.2001May24;411(6836):494-8.doi:10.1038 / 35078107.PMID:11373684. 2.Sun Xiangao,Rogoff Harry A,Li Chiang J.Asymmetric RNA duplexesmediate RNAinterference in mammalian cells.Nat Biotechnol.2008Dec;26(12):1379-82.doi:10.1038 / nbt.1512.Epub 2008Nov 23.Erratumin:NatBiotechnol.2009Feb;27(2):205.PMID:19029911. 3.C.Frank Bennett and Eric E.Swayze,RNA Targeting Therapeutics:Molecular Mechanisms ofAntisense Oligonucleotides as a Therapeutic Platform.Annu.Rev.Pharmacol.Toxicol.2010.50:259–93. 4.C.Frank Bennett.Therapeutic Antisense Oligonucleotides Are ComingofAge.Annu Rev Med.2019Jan 27;70:307-321.doi:10.1146 / annurev-med-041217-010829.PMID:30691367. 5.Setten RL,Rossi JJ,Han SP.The current state andfuture directionsofRNAi-based therapeutics.Nat Rev Drug Discov.2019Jun;18(6):421-446.doi:10.1038 / s41573-019-0017-4.Erratumin:Nat Rev DrugDiscov.2019Mar 18;:Erratumin:Nat Rev DrugDiscov.2019Apr 24;:PMID:30846871. 6.Sibley CR,Seow Y,Wood MJ.Novel RNA-basedstrategiesfor therapeuticgene silencing.MolTher.2010Mar;18(3):466-76.doi:10.1038 / mt.2009.306.Epub2010Jan 19.PMID:20087319;PMCID:PMC2839433. 7.Grimm D.Asymmetry in siRNA design.Gene Ther.2009Jul;16(7):827-9.doi:10.1038 / gt.2009.45.Epub 2009Apr 30.PMID:19404320. 8.Crooke ST,Witztum JL,Bennett CF,Baker BF.RNA-TargetedTherapeutics.Cell Metab.2018Apr 3;27(4):714-739.doi:10.1016 / j.cmet.2018.03.004.Erratumin:Cell Metab.2019Feb5;29(2):501.PMID:29617640. 9.Roberts TC,Langer R,Wood MJA.Advances in oligonucleotide drugdelivery.Nat Rev DrugDiscov.2020Oct;19(10):673-694.doi:10.1038 / s41573-020-0075-7.Epub 2020Aug 11.PMID: 32782413;PMCID:PMC7419031. 10.Ryszard Kole,Adrian R.Krainer,Sidney Altman,RNA therapeutics:Beyond RNA interferenceand antisense oligonucleotids.NatRevDrugDiscov.2016.11(2):125-140. 11.CyA.Stein,Daniela Castanotto,FDA-Approved OligonucleotideTherapies in 2017.MolecularTherapy.2017.Vol.25 No 5 May 2017 12.Richard G.Lee,JeffCrosby,Brenda F.Baker,Mark J.Graham,RosanneM.Crooke,AntisenseTechnology:An EmergingPlatformfor CardiovascularDisease Therapeutics.J.ofCardiovasc.Trans.Res.2013.DOI 10.1007 / s12265-013-9495-7 13.Zamecnik,P.C.,&Stephenson,M.L.Inhibition ofRous sarcoma virusreplication and celltransformation by a specific oligodeoxynucleotide.Proceedings of the National Academy ofSciences USA 75,1978.280–284. 14.Stanley T.Crooke,Molecular Mechanisms ofAntisenseOligonucleotides.NUCLEIC ACIDTHERAPEUTICS.Volume 27,Number 2,2017 Mary AnnLiebert,Inc.DOI:10.1089 / nat.2016.0656 15.Antisense Drug Technologies:Principles,Strategies,andApplications.2.Crooke,ST.,editor.CRC Press;Boca Raton,Florida:2008 16.Fire,A.,Xu,S.,Montgomery,M.K.,Kostas,S.A.,Driver,S.E.,and Mello,C.C.Potent andspecific genetic interference by double-strandedRNA inCaenorhabditis elegans.Nature.1998.391,806–811 17.de Fougerolles A,Vornlocher HP,Maraganore J,LiebermanJ.Interfering with disease:aprogress report on siRNA-basedtherapeutics.Nature Rev Drug Discov.2007;6:443–453.[PubMed:17541417] 18.Jackson AL,Bartz SR,Schelter JM,Kobayashi SV,Burchard J,etal.2003.Expressionprofiling reveals off-targetgene regulationbyRNAi.Nat.Biotechnol.21:635–37 19.Lin X,Ruan X,Anderson MG,McDowell JA,Kroeger P,et al.2005.siRNA-mediatedoff-targetgene silencing triggered by a 7ntcomplementation.NucleicAcids Res.33:4527–35 20.Kwoh JT.2008.An overview ofthe clinical safety experience offirst-and second-generationantisense oligonucleotides.See Ref.9,pp.365–99 21.Henry SP,Kim T-W,Kramer-Strickland K,Zanardi TA,Fey RA,LevinAA.2008. Toxicological properties of 2’-O-methoxyethyl chimeric antisenseinhibitors in animals andman.See Ref.9,pp.327–63 22.Geary,RS.;Yu,RZ.;Levin,AA.Antisense Drug Technologies:Principles,Strategies,andApplications.See Ref.9,pp.183-217 23.Iwamoto N,Butler D,Syrzikapa N,Mohapatra S.,Verdine GL.Control ofphosphorothioatestereochemistry substantially increases the efficacy ofantisense oligonucleotides NatBiotechnol 35(9):845-851,2017 doi:10.1038 / nbt.3948.Epub 2017Aug 21.

Claims

1. An asymmetric short duplex RNA (asdRNA) molecule comprising a first strand and a second strand, wherein the first strand and the second strand both comprise linked nucleotide monomers, wherein the second strand is shorter than the first strand; wherein the first strand is substantially complementary to a target segment of a target RNA through at least one targeting region; wherein the second strand is substantially complementary to the first strand and forms at least one double-stranded region with the first strand; and in, comprising at least one spacer deoxynucleomonomer segment (ISD) in the first strand, the second strand, or both strands of the asdRNA molecule, the ISD comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deoxynucleomonomers; and wherein the total number of deoxynucleotide monomers in the asdRNA molecule does not exceed the total number of ribonucleotide monomers in the asdRNA molecule.

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

3. The asdRNA molecule of claim 2, wherein the at least one improved gene regulatory property or pharmaceutical property comprises: (a) triggering gene silencing at picomolar concentrations, such as 500 pM, 300 pM, 200 pM, 100 pM or lower; (b) Gene silencing is achieved in the cytoplasm as well as in the nucleus and mitochondria; (c) eliminate or reduce interference with endogenous microRNA function, or (d) A wider range of chemical modifications can be tolerated, including non-RNA-like nucleotide modifications or substitutions (e) Lower synthesis cost and improved stability.

4. The asdRNA molecule of claim 1 , wherein at least one ISD comprises at least 4 consecutive deoxynucleoside monomers.

5. The asdRNA molecule of claim 1, wherein at least one ISD is distributed in at least one targeting region of the first strand.

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

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

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

9. The asdRNA molecule of any one of claims 1-8, wherein at least one ISD is distributed in at least one targeting region of the first strand and at least one double-stranded region of the second strand.

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

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 nucleomonomers.

12. The asdRNA molecule of claim 9 or 10, wherein the first strand has a length selected from the group consisting of: a) 8-50 nucleotide monomers, b) 10-36 nucleotide monomers, c) 12-36 nucleotide monomers, and d) 12-25 nucleotide monomers.

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

14. The asdRNA molecule of claim 13, wherein (a) the second strand is shorter than the first strand by at least 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 monomers; (b) the length of the second strand is 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 nucleomonomers; (c) the second strand has a length any number of nucleotide monomers less than the first strand, provided it is capable of forming a duplex with the first strand; and / or (d) at least one of the first base and the last base of the second strand is complementary to a nucleobase in the first strand.

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

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

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

18. The asdRNA molecule of claim 16, wherein the 5'-overhang of the first strand has a length selected from the group consisting of: 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 nucleomonomers, b) 1-15 nucleotide monomers, c) 1-10 nucleotide monomers, d) 1-8 nucleotide monomers, and e) 1-5 nucleotide monomers.

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

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

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

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

23. 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, wherein each internucleoside linkage of the first strand and / or the second strand is a phosphorothioate internucleoside linkage.

24. The asdRNA molecule of claim 20, wherein the modified nucleotide or nucleotide analog comprises a modified sugar moiety, wherein: (a) the 2' position of the modified sugar moiety is substituted with a group selected from the group consisting of OR, R, halogen, SH, SR, NH2, NHR, NR2 and CN, wherein each R is independently C1-C6 alkyl, alkenyl or alkynyl, and halogen is F, Cl, Br or I; (b) the 2' position of the modified sugar moiety is substituted with a group selected from the group consisting of allyl, amino, azido, thio, O-allyl, O-C1-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 ) and O-CH2-C(=O)-N(R1)-(CH2)2-N(R m )(R n ), where each R1, R m and R n are independently H, or substituted or unsubstituted C1-C 10 alkyl. (c) the modified sugar moiety is selected from the group consisting of 5'-vinyl, 5'methyl (R or S), 4'-S, 2'-F, 2'-OCH3, 2'-OCH2CH3, 2'-OCH2CH2F and 2'-O(CH2)2OCH3 substituents. (d) wherein the modified sugar moiety is substituted with a bicyclic sugar selected from the group consisting 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(CH2OCH3)—O-2′, 4′-C(CH3)(CH3)—O-2′, 4′-CH2—N(OCH3)-2′, 4′-CH2—O—N(CH3)-2′, 4′-CH2—N(R)—O-2′ (wherein R is H, C1-C 12 alkyl or protecting group), 4′-CH2—C(H)(CH3)-2′, and 4′-CH2—C—(═CH2)-2′, and / or (e) The modified sugar moiety is selected from the group consisting of: 2'-O-methoxyethyl modified sugar (MOE), 4'-(CH2)-O-2' bicyclic sugar (LNA), 2'-deoxy-2'-fluoroarabinose (FANA) and methyl(methyleneoxy)(4'-CH(CH3)-O-2) bicyclic sugar (cEt).

25. The asdRNA molecule of any one of claims 1-19, 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 asdRNA molecule of claim 20, wherein the modified nucleotide or nucleotide analog comprises a modified nucleobase, wherein: (a) The modified nucleobase is selected from the group consisting of: 5-methylcytosine (5-Me-C), hypoxanthine nucleobases, 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-pseudouracil, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3) uracil and cytosine and pyrimidine bases. Other alkynyl derivatives, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo (especially 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 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 5-methylcytosine.

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

28. The asdRNA molecule of claim 1, wherein the targeting RNA is mRNA, pre-mRNA, mt-RNA or non-coding RNA, wherein these RNAs encode proteins associated with the disease or regulate part of a biological pathway associated with the disease.

29. The asdRNA molecule of claim 1, wherein the target RNA is selected from the group consisting of: a) mRNA, pre-mRNA or mt-RNA of a gene associated with a disease or condition in humans or animals, b) mRNA or pre-mRNA of genes of pathogenic microorganisms, c) Viral RNA d) lncRNA, e) miRNA, and f) RNA associated with 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, skin diseases, cachexia, gastrointestinal diseases, respiratory disorders, cardiovascular disorders, renal diseases, rheumatoid diseases, nervous system disorders, endocrine disorders, and diseases associated with aging.

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

31. The asdRNA molecule of claim 30, wherein the ligand or moiety is selected from the group consisting of a polypeptide / protein, 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 a nucleic acid aptamer.

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

33. The pharmaceutical composition of claim 32, wherein the carrier is selected from the group consisting of a drug 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. A method of treating or preventing a disease or condition, wherein the method comprises administering to a subject in need thereof a therapeutically effective dose of the asdRNA molecule of any one of claims 1-31 or the pharmaceutical composition of claim 32 or 33.

35. The method of claim 34, wherein the disease or condition is selected from the group consisting of cancer, autoimmune diseases, inflammatory diseases, degenerative diseases, infectious diseases, proliferative diseases, metabolic diseases, immune-mediated disorders, allergic diseases, skin diseases, malignancies, gastrointestinal diseases, liver diseases, respiratory disorders, cardiovascular disorders, skin diseases, kidney diseases, rheumatoid diseases, nervous system disorders, psychiatric disorders, endocrine disorders, and disorders or diseases associated with aging.

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), oral administration (po), intramuscular injection (im), oral administration, inhalation, topical, intrathecal, and other sites of administration.

37. A method of modulating gene expression or function in a eukaryotic cell, wherein the method comprises contacting the cell with an effective amount of the asdRNA molecule of any one of claims 1-31 or the pharmaceutical composition of claim 32 or 33.

38. An asymmetric short duplex RNA (asdRNA) molecule comprising a first strand and a second strand, wherein both the first strand and the second strand comprise linked nucleomonomers, wherein the first strand comprises at least one spacer deoxynucleomonomer (ISD), wherein the nucleotide monomers are selected from the group consisting of naturally occurring ribonucleotides, their analogs and modified ribonucleotides; wherein the deoxynucleotide monomers are selected from the group consisting of naturally occurring deoxynucleotides, their analogs and modified deoxynucleotides; wherein at least one ISD comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive deoxynucleotide monomers; wherein the second chain is shorter than the first chain by a number of monomers selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 monomers; wherein the first strand is substantially complementary to a target segment of a target RNA through at least one targeting region, wherein the first strand consists of 10-36 (both end points of the range are inclusive) nucleoside monomers connected by bonds, wherein the bonds are selected from the group consisting of phosphorothioate bonds, phosphodiester bonds, or a mixture of phosphorothioate and phosphodiester bonds between adjacent monomers, wherein the second strand is substantially complementary to the first strand and forms at least one double-stranded region with the first strand, wherein the second strand consists of 8-32 (both end points of the range are inclusive) nucleoside monomers connected by bonds selected from the group consisting of phosphorothioate bonds, phosphodiester bonds, or a mixture of phosphorothioate and phosphodiester bonds between adjacent monomers, and The total number of deoxynucleotide monomers in the asdRNA molecule does not exceed the total number of ribonucleotide monomers in the asdRNA molecule.

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