Short double-stranded RNA (Ribonucleic Acid) with deoxyribonucleotide spacer fragment as gene silencing technology and application of 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 a short duplex RNA molecule, wherein the length of the sense strand is at least equal to the length of the antisense strand. The duplex RNA molecule further comprises at least one deoxynucleotide 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 the benefit of U.S. Provisional Patent Application No. 63 / 431,307, filed 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 short double-stranded RNAs with deoxyribonucleotide spacer segments, and related compositions and methods thereof, useful for biological or medical research, disease treatment and prevention, and gene silencing applications in other biological fields. BACKGROUND

[0003] Modern medical therapies rely on two fundamental technologies, namely small molecule chemistry and protein / antibody technologies. However, only about 10% of the targets identified by genomics research and biomedical institutes can be addressed by the two fundamental technologies. Oligonucleotides are promising to address numerous targets, including targets that are undruggable by small molecule chemistry and protein / antibody technologies. Antisense oligonucleotide (ASO) and small interfering RNA (siRNA) technologies have been created over 40 years of research (Cy A. Stein et al., 2017). However, despite 40 years of research, significant drugability issues have hindered the development of ASO and siRNA technologies as a mainstream therapeutic platform, except for a few clinical orphan indications. These drugability issues include, among others: low silencing efficiency, off-target effects, stimulation of unintended immune responses, tissue penetration challenges, and in vivo delivery, etc. Therefore, there is a significant unmet need to create new technologies to target genes of interest in various biological and medical applications.

[0004] ASO is a gene silencing technology based on a concept first proposed in 1978 (Zamecnik P.C. et al., 1978). In general, the principle behind the ASO technology is that antisense oligonucleotides hybridize to the target nucleic acid, modulating the activity or function of gene expression, such as transcription / post-transcription or translation. Its mechanisms are broadly classified as: (1) occupancy without promoting degradation of RNA, in which binding of ASO leads to translational arrest, splicing inhibition or induction of alternative splicing variants, or (2) occupancy-induced destabilization, in which binding of ASO promotes degradation of RNA by endogenous enzymes, such as ribonuclease H1 (RNase H1); and (3) translational modulation: ASO can block upstream open reading frames (uORFs) or other inhibitory or regulatory elements in the 5’UTR region, increasing or modulating translation efficiency (Stanley T. Crooke et al., 2008; C. Frank Bennett, 2010; Richard G. Lee, 2013; Stanley T. Crooke, 2017). ASO is a structure of single-stranded deoxyribonucleotide sequence that can bind to the target RNA through base pairing. Through 40 years of research, ASO technology has been improved by various chemical modifications to single-stranded oligonucleotides, such as phosphorothioate substitution or other modified nucleotides (see Iwamoto N et al 2017, Crooke ST, 2017; Crooke ST et al., 2018; U.S. Pat. Nos. 7919472 and 9045754).

[0005] Short double-stranded RNA triggers homologous sequence RNA loss through the RNAi mechanism, which was first observed in plants and later confirmed in nematodes (Caenorhabditis elegans) (A. Fire et al, 1998). This mechanism involves the degradation of long dsRNA into short interfering duplex RNA (siRNA), which interacts with the multi-protein RNA-induced silencing complex (RICS). In RISC, the siRNA is unwound, with the sense strand discarded and the antisense or guide strand bound to the RISC endonuclease AG02, which subsequently cleaves the target RNA (de Fougerolles et al., 2007; Ryszard Kole, 2016). RNAi is a sequence-specific post-transcriptional gene silencing process triggered by short double-stranded RNA in the cytoplasm. In mammalian cells, synthetic siRNA or asymmetric short interfering RNAs (aiRNA or asymmetric siRNA) can be used to induce gene silencing through the RISC-dependent RNAi mechanism (see Elbashir SM et al., 2001; Sun X et al., 2008; U.S. Pat. Nos. 7056704 and 9328345).

[0006] Oligonucleotides have been studied for decades and are considered promising as a new class of therapeutics. However, their limited silencing efficiency, delivery challenges, and dose-dependent side effects (including hybridization-dependent and hybridization-independent toxicities) have consistently limited the development of these novel therapeutics (C. Frank Bennett, 2010; C. Frank Bennett, 2019; Roberts TC et al., 2020; Crooke ST et al., 2018; and Setten RL et al., 2020). In general, while ASO compounds are less potent than siRNA-based compounds in inducing gene silencing, ASO compounds have some pharmacological advantages over siRNA compounds. Currently, ASO and siRNA remain two equally important platform technologies for designing gene silencing therapeutic treatments (Crooke ST et al 2018; Roberts TC et al 2020). Hybridization-dependent toxicity of oligonucleotides is mainly attributed to their hybridization with non-target genes (“off-target effects”) (Jackson et al., 2003; Lin X et al., 2005). Hybridization-independent toxicity of oligonucleotides occurs through their interactions with proteins: these interactions include increasing clotting time, pro-inflammatory 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 tubular lesions and thrombocytopenia (Geary, RS. et al., 2007; Kwoh J T, 2008). Clinically, the major tolerability and safety issues of the first generation PS antisense oligodeoxynucleotides and the second generation 2’-MOE modified antisense oligonucleotides have been shown to be hybridization-independent effects, such as prolonging 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 much less potent than siRNAs and have been shown to have dose-dependent, typical toxicities (Kendall S. Frazier, 2015). Over the past 40 years, efforts have been made to overcome the limited potency and related safety issues of ASOs through various chemical modifications to mitigate dose-dependent toxicities of oligonucleotides (Iwamoto N et al 2017, Crooke ST et al., 2018; and Roberts TC et al., 2020).

[0007] The off-target silencing effect of siRNA duplexes is thought to be mediated by sense strand-mediated silencing, competition with endogenous miRNA pathways, and interactions with TLR or other proteins, compared to ASOs (Setten RL et al 2019). In addition, typical 21 nt / 19 bp siRNA duplexes are not efficient in cell and tissue penetration and require extensive chemical modifications to enhance the stability of siRNA and other pharmaceutical properties.

[0008] In summary, after 40 years of ASO technology innovation and 20 years of RNAi-based technology research, it is still challenging to successfully develop a gene-targeting therapy for nearly 90% of the target points associated with human diseases. In addition, for the currently approved oligonucleotide drugs, the cost per patient per year exceeds $500,000, so it cannot solve the diseases affecting the general population. Therefore, there is an urgent need for new technologies to overcome these challenges.

[0009] The references cited herein are not to be considered an admission that the present invention, as claimed, is SUMMARY

[0010] The present invention is based on the unexpected discovery of efficient gene silencing triggered by short duplex ribonucleotides (sdRNA) having interspersed segments of deoxyribonucleotides (ISD). This novel gene silencing technology is achieved by sdRNA having one or more interspersed segments of deoxyribonucleotides, which employs a short duplex molecule consisting of linked nucleotide monomers, wherein each nucleotide monomer is selected from the group consisting of naturally occurring nucleotides, analogs thereof, 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, analogs thereof, and modified ribonucleotides. Further, by incorporating one or several interspersed segments of deoxyribonucleotide monomers, the gene silencing function of sdRNA can be significantly achieved or enhanced. The "deoxyribonucleotide monomers" can 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 sdRNA molecule of the present invention are ribonucleotide monomers, thus, the entire molecule is referred to as a double-stranded RNA molecule, or more specifically, a short double-stranded RNA (sdRNA) molecule. The molecule of the present invention is further spaced apart by deoxyribonucleotide monomers to form at least one deoxyribonucleotide monomer interval segment (ISD).

[0012] In one embodiment, the powerful gene silencing effect of the novel platform technology of sdRNA-based of the present disclosure and other advantages disclosed below are achieved by an oligonucleotide monomer sense strand and an oligonucleotide monomer antisense strand, wherein the oligonucleotide monomer antisense strand is substantially complementary to a target ribonucleotide sequence. Our data show that the sdRNA molecule of the present invention, due to its unique and novel composition, can cause gene silencing at picomolar (e.g., 800 pM, 500 pM, 300 pM, 200 pM, 100 pM or lower concentration) concentration, which is more potent than existing gene silencing technologies, thus, can reduce dose-dependent toxicity. The sdRNA molecule of the present invention also has at least one of the following advantages over existing gene silencing technologies, including that, unlike siRNA-based gene silencing, which only occurs in the cytoplasm, sdRNA can achieve gene silencing in the cytoplasm, as well as in the nucleus and mitochondria, etc.; reduce off-target effects; eliminate or reduce the undesirable interference with endogenous microRNA function present in siRNA; better tissue penetration; better stability; low synthesis cost and improved pharmaceutical properties. Thus, the sdRNA molecule of the present invention has great potential in addressing various challenges faced by existing gene silencing technologies. In addition, the sdRNA molecule of the present invention can modulate gene expression activity or function at the transcription stage, post-transcriptional stage and / or translation stage, while RNAi can only trigger gene silencing at the post-transcriptional level. In addition, sdRNA can allow more and wider chemical modifications, including non-RNA-like nucleotide modifications or substitutions. The sdRNA molecule of the present invention can be used in all fields in which current oligonucleotides are being applied or are expected to be used, including research, diagnosis, disease prevention and treatment, and other applications in the biological field, also including the fields of pesticides and veterinary drugs.

[0013] In a first aspect, the present invention provides a composition comprising a short duplex RNA (sdRNA) molecule having a first strand and a second strand, wherein each of the first strand and the second strand comprises linked ribonucleotide monomers and deoxyribonucleotide monomer spacer segments. The ribonucleotide monomers in the sdRNA molecule are selected from the group consisting of naturally occurring ribonucleotides, analogs thereof, and modified ribonucleotides; the deoxyribonucleotide monomer spacer segments in the sdRNA molecule are selected from the group consisting of naturally occurring deoxyribonucleotides, analogs thereof, and modified deoxyribonucleotides. The second strand is at least as long as the first strand, more specifically, the second strand is equal in length to the first strand or longer than the first strand. The first strand can be considered an antisense strand or antisense oligonucleotide due to its substantial complementarity to a target segment of a target RNA via at least one targeting region. Further, the second strand, which can be considered a sense strand or sense oligonucleotide, is substantially complementary to the first strand and forms at least one duplex region with the first strand. The sdRNA molecule comprises at least one deoxyribonucleotide monomer spacer segment (ISD) having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deoxyribonucleotide monomers, wherein the ISD can be present in either strand or in both strands. The total number of deoxyribonucleotide monomers in the sdRNA molecule of the present invention does not exceed the total number of ribonucleotide monomers in any given sdRNA molecule.

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

[0015] In one feature, the sdRNA containing the ISD has better gene regulation or drug properties compared to the corresponding RNA duplex without the ISD. In other words, the sdRNA of the present disclosure containing at least one ISD has better or more desirable at least one gene regulation or drug property; the property is selected from the group consisting of: ability to function at subcellular locations, potential target RNA of interest, potency, efficacy, speed of onset, durability, synthetic economy, off-target effects, available chemical modifications, non-specific immune stimulation, stability, and delivery. More specifically, the improved gene regulation or drug property of the sdRNA molecule of the present disclosure compared to the corresponding RNA duplex without the ISD means, for example, that one or more of the following can be achieved: the ability to function in gene regulation not only in the cytoplasm of the cell, 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 (mitochondrion messenger RNA)); better potency and / or efficacy; faster onset; improved pharmacokinetic properties; longer durability; fewer off-target effects; less typical toxicity of dose-dependence; avoidance of non-specific interferon-like responses; and lower manufacturing cost; more allowable / possessable chemical modifications (including non-RNA-like nucleotide modifications or substitutions), better stability, and better delivery. The corresponding RNA duplex refers to a short duplex RNA molecule without the ISD of the present disclosure, wherein the antisense strand targets the same or substantially the same sequence as the sdRNA. More specifically, the sdRNA with the ISD can also be used to target or silence RNA in the nucleus, such as pre-mRNA, non-coding RNA, and long non-coding RNA, as well as to target or silence RNA in the mitochondria, such as mt-mRNA (mitochondrion messenger RNA), whereas RNAi technology (such as siRNA with short double-stranded RNA structure) can only function in the cytoplasm. Thus, the sdRNA with the ISD can target more target RNAs, disease-causing genes, and have a broader application prospect compared to existing gene silencing technologies, especially RISC-dependent RNAi gene silencing technologies. In another feature, the sdRNA molecule of the present disclosure has better gene regulation or drug properties compared to the corresponding single-stranded antisense oligonucleotide (ASO). In other words, the sdRNA has at least one gene regulation or drug property that is better or more desirable than the corresponding ASO; the property is selected from the group consisting of: potency, efficacy, speed of onset, durability, synthetic economy, off-target effects, non-specific immune stimulation, stability, and delivery. The corresponding antisense oligonucleotide refers to a single-stranded antisense oligonucleotide that targets the same or substantially the same sequence as the at least one targeting region in the first strand of the sdRNA molecule.

[0016] The compositions provided herein are used to modulate gene expression or function in a eukaryotic cell, wherein the sdRNA is contacted with the cell or administered to a subject.

[0017] In one feature, the first strand of the sdRNA molecule comprises at least one ISD, and / or the second strand can comprise at least one ISD. In one embodiment, the first strand of the sdRNA molecule comprises at least one ISD, and the second strand also comprises at least one ISD. In one feature, the at least one ISD is distributed in at least one targeting region of the first strand, and the at least one ISD is distributed in at least one double-stranded region of the second strand. In one embodiment, the first strand of the sdRNA molecule comprises at least one ISD, and the second strand consists of ribonucleotide monomers.

[0018] In one feature, each ISD independently consists of one deoxynucleotide monomer, or comprises at least 2, 3, 4, 5, or more contiguous deoxynucleotide monomers. In one embodiment, the at least one ISD comprises at least 4 contiguous deoxynucleotide monomers. In another feature, the ISD comprises at least 2 deoxynucleotide monomers, wherein the deoxynucleotide monomers are contiguous or are separated by at least one (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) different kind of monomer. In one feature, the ISD is separated by at least 2 (2, 3, 4, 5, 6, 7, 8, 9, 10, or more) different kind of monomers.

[0019] In one embodiment, the at least one ISD is distributed in the first strand. In one feature, the at least one ISD is distributed in at least one targeting region of the first strand. In various embodiments, the at least one ISD in the targeting region of the first strand comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous deoxynucleotide monomers. In different embodiments, the at least one ISD in the first strand comprises at least 4 or 5 contiguous deoxynucleotide monomers. In different embodiments, the at least one ISD in the targeting region of the first strand comprises at least 4 or 5 contiguous deoxynucleotide monomers. In one embodiment, there is only one ISD in the first strand, wherein the ISD comprises at least 4 or 5 contiguous deoxynucleotide monomers. In another embodiment, there are two or more ISDs in the first strand, wherein each ISD independently consists of one deoxynucleotide monomer, or comprises at least 2, 3, 4, 5, or more contiguous deoxynucleotide monomers. In another embodiment, there are two or more ISDs in the first strand, wherein one ISD comprises at least 4 or 5 contiguous deoxynucleotide monomers, and the other ISDs independently consist of one deoxynucleotide monomer, or comprise at least 2, 3, 4, 5, or more contiguous deoxynucleotide monomers.

[0020] In one embodiment, at least one ISD is distributed in the second strand. In another feature, at least one ISD is distributed in at least one double-stranded region of the second (sense) 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 contiguous deoxynucleotide monomers. In another feature, an ISD in the second strand comprises at least 2 deoxynucleotide monomers, wherein the deoxynucleotide monomers are contiguous or are separated by at least one (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) different kind of monomer.

[0021] In some embodiments, the ISD is distributed in a more central portion of the first strand (at least 1, 2, 3, 4, or 5 nucleotides from the ends of the strand, i.e., the 2ndposition or more central position from the end of the strand). In some embodiments, the ISD can be distributed anywhere in the second strand. In some embodiments, the ISD is distributed in a more central portion of the second strand (at least 1, 2, 3, 4, or 5 nucleotides from the ends of the strand, i.e., the 2ndposition or more central position from the end of the strand). In some embodiments, at least one end of the first strand and / or the second strand (i.e., the first nucleotide monomer from the 3' end, the 5' end, or both ends) is not a deoxynucleotide monomer. In some embodiments, the first nucleotide monomer from the 3' end of the first strand and / or the second strand is not a deoxynucleotide monomer.

[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 the group consisting of mRNA, pre-mRNA, mt-mRNA, and non-coding RNA, wherein the RNA either encodes a protein associated with a disease or modulates a portion of a biological pathway associated with a disease, e.g., a mammalian disease.

[0023] In various embodiments, the first strand has a backbone length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 linked nucleotide monomers, or an equivalent length thereof, or a length range bracketed by any two of the aforementioned values (both endpoints of the range are included). For example, some length ranges for the first strand (antisense strand) include (a) 8-36 nucleotide monomers; (b) 8-33 nucleotide monomers; (c) 10-30 nucleotide monomers; (d) 10-29 nucleotide monomers; (e) 12-29 nucleotide monomers; (f) 12-28 nucleotide monomers; (g) 12-26 nucleotide monomers; (h) 12-25 nucleotide monomers; (i) 13-25 nucleotide monomers; (j) 13-24 nucleotide monomers; (k) 13-23 nucleotide monomers; (1) 14-24 nucleotide monomers; (m) 15-23 nucleotide monomers; and (n) 8-50 nucleotide monomers; (o) 16-23 nucleotide monomers; (p) 10-36 nucleotide monomers; and (r) at least 8 nucleotide monomers. In certain embodiments, when the backbone length of the first strand is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 linked nucleotide monomers, or an equivalent length thereof, at least one ISD can be distributed at any position of the first strand, and if present in the second strand, at any position of 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. Thus, the two strands form a double-stranded region comprising 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 base pairs. 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, 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 a nucleobase selected from the group consisting of A, G, C, U, and T, or from a modified nucleobase.

[0025] In one feature, the second strand has a backbone length equal to the backbone length of the first strand. In one embodiment, the two strands of the sdRNA molecule form a symmetric duplex without any overhangs. In another embodiment, both the first strand and the second strand have a 3' overhang or a 5' overhang. In certain embodiments, the 3' overhang or 5' overhang of the two strands is at least 1, 2, 3, 4, or 5 nucleotide monomers in length. In certain embodiments, the 3' overhang or 5' overhang of the two strands is the same length and is at least 1, 2, 3, 4, or 5 nucleotide monomers in length.

[0026] In another feature, the second strand has a backbone length that is at least longer than the first strand by a number of nucleotide monomers that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, the 3' overhang of the second strand 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, or 30 nucleotide monomers in length, or a length range bracketed by any two of the aforementioned values (both of the range's endpoints are included). In certain embodiments, the 5' overhang of the second strand 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, or 30 nucleotide monomers in length, or a length range bracketed by any two of the aforementioned values (both of the range's endpoints are included). In one embodiment of the application, the second strand has a 3' overhang of 1-10 nucleotide monomers and a 5' overhang of 1-10 nucleotide monomers (both of the range's endpoints are included). In another embodiment, the second strand has a 3' overhang of 1-26 nucleotide monomers (both of the range's endpoints are included) and a 5' overhang of 1-26 nucleotide monomers (both of the range's endpoints are included). In another embodiment, the second strand has a 3' overhang of 1-8 nucleotide monomers (both of the range's endpoints are included) and a 5' overhang of 1-8 nucleotide monomers (both of the range's endpoints are included). In another embodiment, the second strand has a 3' overhang of 9-26 nucleotide monomers (both of the range's endpoints are included) and a 5' overhang of 9-26 nucleotide monomers (both of the range's endpoints are included). In another embodiment, the second strand has a 3' overhang of 14-26 nucleotide monomers (both of the range's endpoints are included) and a 5' overhang of 14-26 nucleotide monomers (both of the range's endpoints are included). In another embodiment, the second strand has a 5' overhang of 1-26 nucleotide monomers (both of the range's endpoints are included) and a 3' overhang of 1-26 nucleotide monomers (both of the range's endpoints are included). In another embodiment, the second strand has a 5' overhang of 1-8 nucleotide monomers (both of the range's endpoints are included) and a 3' overhang of 1-8 nucleotide monomers (both of the range's endpoints are included). In another embodiment, the second strand has a 5' overhang of 9-26 nucleotide monomers (both of the range's endpoints are included) and a 3' overhang of 9-26 nucleotide monomers (both of the range's endpoints are included). In another embodiment, the second strand has a 5' overhang of 14-26 nucleotide monomers (both of the range's endpoints are included) and a 3' overhang of 14-26 nucleotide monomers (both of the range's endpoints are included). In another embodiment, the second strand has a 3' overhang of 1-10 nucleotide monomers (both of the range's endpoints are included) and a 5' recessed end of 1-10 nucleotide monomers (both of the range's endpoints are included), or the second strand has a 5' overhang of 1-10 nucleotide monomers (both of the range's endpoints are included) and a 3' recessed end of 1-10 nucleotide monomers (both of the range's endpoints are included).

[0027] In various embodiments, the second strand has a backbone length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 linked nucleotide monomers, or an equivalent length, or a length range bracketed by any two of the aforementioned values (both endpoints of the range are included). For example, in certain embodiments, some length ranges for the second / sense strand include: (a) 8-36 nucleotide monomers; (b) 8-33 nucleotide monomers; (c) 10-30 nucleotide monomers; (d) 10-29 nucleotide monomers; (e) 12-29 nucleotide monomers; (f) 12-28 nucleotide monomers; (g) 12-26 nucleotide monomers; (h) 12-25 nucleotide monomers; (i) 12-24 nucleotide monomers; (j) 13-25 nucleotide monomers; (k) 13-24 nucleotide monomers; (1) 13-23 nucleotide monomers; (m) 14-24 nucleotide monomers; (n) 15-23 nucleotide monomers; (o) 8-50 nucleotide monomers; (p) 16-23 nucleotide monomers; (q) 10-36 nucleotide monomers; and (r) at least 8 nucleotide monomers. In certain embodiments, where the second strand is capable of forming a thermodynamically stable duplex with the first strand, the backbone length of the second strand can have any number of nucleotide monomers greater than the length of the first strand.

[0028] In one feature of the sdRNA molecules of the application, at least one nucleotide monomer in the first strand and / or the 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 includes a phosphorothioate group (P=S), a phosphotriester, a methylphosphonate, or an aminophosphonate.

[0029] In certain embodiments, 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. In some embodiments, each internucleoside linkage of the first strand and / or the second strand is a phosphorothioate internucleoside linkage. In various embodiments, the internucleoside linkages of the first strand and / or the second strand are a mixture of phosphorothioate linkages and phosphodiester linkages. In certain embodiments, the internucleoside linkages between each deoxynucleoside monomer in the first strand and / or the second strand are naturally occurring internucleoside linkages or a mixture of phosphorothioate and phosphodiester linkages. In certain embodiments, at least one internucleoside linkage between deoxynucleotide monomers in the second strand is a phosphorothioate internucleoside linkage. In certain embodiments, all of the nucleoside monomers of the second strand of the sdRNA molecule are linked by naturally occurring internucleoside linkages or modified internucleoside linkages, such as phosphorothioate internucleoside linkages. In some embodiments, all of the ribonucleoside monomers of the second strand of the sdRNA molecule are linked by naturally occurring internucleoside linkages or phosphorothioate internucleoside linkages. In some embodiments, at least one internucleoside linkage between nucleoside monomers located in a more central portion of the second strand, such as position 4 or more central positions counting from both ends, is a modified internucleoside linkage, such as a phosphorothioate internucleoside linkage.

[0030] In some embodiments, each nucleoside monomer of the first strand of the sdRNA molecule is linked by a naturally occurring internucleoside linkage. In some embodiments, each ribonucleoside monomer of the first strand of the sdRNA molecule is linked by a naturally occurring internucleoside linkage.

[0031] In one feature, the first strand and / or the second strand of the molecules of the present application comprises 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 with a group selected from the group consisting of OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein each R is independently a 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 with a group selected from the group consisting of allyl, amino, azido, thio, O-allyl, O-C1-C6 alkyl, OC1-C6 alkenyl, OC1-C6 alkynyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-O-N(R1)(R2), O-CH2-C(=O)-N(R1)(R2), or O-CH2-C(=O)-N(R1)-(CH2)2-N(R1)(R2), wherein each R1 and R2 is independently H, C1-C6 alkyl, C1-C6 alkenyl, or C1-C6 alkynyl. 10 alkyl, OC1-C6 alkenyl, OC1-C6 alkynyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-O-N(R1)(R2), O-CH2-C(=O)-N(R1)(R2), or O-CH2-C(=O)-N(R1)-(CH2)2-N(R1)(R2), wherein each R1 and R2 is independently H, C1-C6 alkyl, C1-C6 alkenyl, or C1-C6 alkynyl. m alkyl, OC1-C6 alkenyl, OC1-C6 alkynyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-O-N(R1)(R2), O-CH2-C(=O)-N(R1)(R2), or O-CH2-C(=O)-N(R1)-(CH2)2-N(R1)(R2), wherein each R1 and R2 is independently H, C1-C6 alkyl, C1-C6 alkenyl, or C1-C6 alkynyl. n alkyl, OC1-C6 alkenyl, OC1-C6 alkynyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-O-N(R1)(R2), O-CH2-C(=O)-N(R1)(R2), or O-CH2-C(=O)-N(R1)-(CH2)2-N(R1)(R2), wherein each R1 and R2 is independently H, C1-C6 alkyl, C1-C6 alkenyl, or C1-C6 alkynyl. m alkyl, OC1-C6 alkenyl, OC1-C6 alkynyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-O-N(R1)(R2), O-CH2-C(=O)-N(R1)(R2), or O-CH2-C(=O)-N(R1)-(CH2)2-N(R1)(R2), wherein each R1 and R2 is independently H, C1-C6 alkyl, C1-C6 alkenyl, or C1-C6 alkynyl. n alkyl, OC1-C6 alkenyl, OC1-C6 alkynyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-O-N(R1)(R2), O-CH2-C(=O)-N(R1)(R2), or O-CH2-C(=O)-N(R1)-(CH2)2-N(R1)(R2), wherein each R1 and R2 is independently H, C1-C6 alkyl, C1-C6 alkenyl, or C1-C6 alkynyl. m alkyl, OC1-C6 alkenyl, OC1-C6 alkynyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-O-N(R1)(R2), O-CH2-C(=O)-N(R1)(R2), or O-CH2-C(=O)-N(R1)-(CH2)2-N(R1)(R2), wherein each R1 and R2 is independently H, C1-C6 alkyl, C1-C6 alkenyl, or C1-C6 alkynyl. n alkyl, OC1-C6 alkenyl, OC1-C6 alkynyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-O-N(R1)(R2), O-CH2-C(=O)-N(R1)(R2), or O-CH2-C(=O)-N(R1)-(CH2)2-N(R1)(R2), wherein each R1 and R2 is independently H, C1-C6 alkyl, C1-C6 alkenyl, or C1-C6 alkynyl. mand R n independently H or a substituted or unsubstituted C1-C 10 alkyl. In some embodiments, the modified sugar moiety has a substituent 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'-0(CH2)2OCH3. In some embodiments, the modified sugar moiety is substituted with a bicyclic sugar selected from the group consisting of 4'-(CH2)-0-2' (LNA), 4'-(CH2)-S-2', 4'-(CH2)2-0-2' (ENA), 4'-CH(CH3)-0-2' (cEt), and 4'-CH(CH2OCH3)-0-2', 4'-C(CH3)(CH3)-0-2', 4'-CH2-N(OCH3)-2', 4'-CH2-0-N(CH3)-2', 4'-CH2-N(R)-0-2' (where R is H, C1-C 12 alkyl or a protecting group), 4'-CH2-C(H)(CH3)-2', and 4'-CH2-C(=CH2)-2'. In some embodiments, the modified sugar moiety is selected from the group consisting of 2'-0-methoxyethyl modified sugar (MOE), 4'-(CH2)-0-2' bicyclic sugar (LNA), 2'-deoxy-2'-fluoroarabinose (2'-F arabinose, FANA), and methyl(methyloxy)(4'-CH(CH3)-0-2) bicyclic sugar (cEt).

[0032] In certain embodiments, either all of the nucleoside monomers in the second strand are sugar moiety-unmodified nucleoside monomers, or each nucleoside monomer has a modified sugar moiety. In certain embodiments, either all of the ribonucleoside monomers in the second strand are sugar moiety-unmodified purine ribonucleoside monomers, or each ribonucleoside monomer has a modified sugar moiety, e.g., a 2'-F modified sugar, a 2'-OMe modified sugar, a 2'-0-methoxyethyl modified sugar (MOE), a 4'-(CH2)-0-2' bicyclic sugar (LNA), and a methyl(methyloxy)(4'-CH(CH3)-0-2) bicyclic sugar (cEt). In certain embodiments, at least one of the ribonucleoside monomers distributed in the more central portion of the second strand (e.g., positions 4 or more central from both ends) has a modified sugar moiety, e.g., a 2'-F modified sugar, a 2'-OMe modified sugar, a 2'-0-methoxyethyl modified sugar (MOE), a 4'-(CH2)-0-2' bicyclic sugar (LNA), or a methyl(methyloxy)(4'-CH(CH3)-0-2) bicyclic sugar (cEt).

[0033] In one particular 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 particular embodiment, the ISD can 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 sdRNA molecules of the present application, the sugar moiety of the ribonucleotide monomer is selected from the group consisting of: naturally occurring ribonucleotide (2-OH), 2'-F modified sugar, 2'-OMe modified sugar, 2'-0-methoxyethyl modified sugar (MOE), 4'-(CH2)—O-2' bicyclic sugar (LNA), and methyl(methyloxy) (4'-CH(CH3)—O-2) bicyclic sugar (cEt).

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

[0037] In another feature, the first strand and / or the second strand of a molecule of the application comprises at least one nucleotide monomer, wherein the nucleotide monomer comprises a modified nucleobase. In some embodiments, the modified nucleobase is selected from the group consisting of 5-methylcytosine (5-Me-C), inosine bases, tritylated bases, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl (-CºC-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 1 -methyl-pseudouracil, 8- halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (especially 5-bromo), 5-trifluoromethyl, 5-methyl uracil and other 5- substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2- aminoadenine, 8-azaguanine and 8-azadenine, 7-deazaguanine and 7-deazaadenine, and 3- deazaguanine and 3-deazaadenine. In particular embodiments, the modified nucleobase is 5-methylcytosine. In one embodiment, every cytosine base of a molecule of the application is 5-methylcytosine. In one embodiment, every uridine base in a ribonucleotide monomer of a sdRNA molecule of the application is 5-methyluridine.

[0038] In one feature, the first strand and / or the second strand of a molecule of the application is conjugated to a ligand or moiety. In certain embodiments, the ligand or moiety is selected from the group consisting of a peptide / 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, a cholesterol, a GalNAc, and an aptamer.

[0039] In one feature of the application, a sdRNA molecule is used to modulate gene expression or function in a cell (e.g., a eukaryotic cell, such as a mammalian cell).

[0040] In certain embodiments, the target RNA, from which at least part of the nucleotide monomer sequence of the sdRNA molecule is determined according to the principles of the present application, is selected from the group consisting of mRNA, pre-mRNA, mt-mRNA, and non-coding RNA. In one feature, these target RNAs either encode proteins associated with a disease or modulate biological pathways associated with a disease. In various embodiments, such target RNAs can be, but are not limited to, selected from the group consisting of: mRNA, pre-mRNA, mt-mRNA, non-coding RNA, or IncRNA of a gene associated with a disease or disorder in a human or animal; mRNA or pre-mRNA of a pathogenic microorganism gene; viral RNA, and RNA associated with a disease or disorder selected from the group consisting of an autoimmune disease, an inflammatory disease, a degenerative disease, an infectious disease, a proliferative disease, a metabolic disease, an immune-mediated disorder, an allergic disease, a skin disease, a malignant disease, a gastrointestinal disease, a respiratory disease, a cardiovascular disease, a renal disease, a rheumatoid disease, a neurological disease, an endocrine disorder, and a disease or disorder associated with aging.

[0041] In one embodiment, the present application provides a short duplex RNA (sdRNA) 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 deoxyribonucleotide monomer Interspersed Segment (ISD), wherein: (a) the first strand is equal in length to the second strand, or is at least one monomer shorter than the second strand, the monomer being selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 monomers; (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 consists of 8-36 (both endpoints of the range included therein) nucleoside monomers linked by bonds, wherein the bonds are selected from the group consisting of phosphorothioate bonds between adjacent monomers, phosphodiester bonds, and a mixture of phosphorothioate bonds and phosphodiester bonds; (c) the second strand is substantially complementary to the first strand, forming at least one double-stranded region with the first strand, wherein the second strand consists of 10-36 (both endpoints of the range included therein) nucleoside monomers linked by bonds, wherein the bonds are selected from the group consisting of phosphorothioate bonds between adjacent monomers, phosphodiester bonds, and a mixture of phosphorothioate bonds and phosphodiester bonds; (d) the 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 sdRNA molecule comprises at least one deoxyribonucleotide monomer, wherein the deoxyribonucleotide monomer is selected from the group consisting of deoxyribonucleotides, analogs thereof, and modified deoxyribonucleotides; and (f) the total number of deoxyribonucleotide monomers in the sdRNA molecule does not exceed the total number of ribonucleotide monomers therein. In one feature, the sdRNA 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 a further feature, the sdRNA molecule silences the expression of the target gene in the cell more strongly or more effectively than a corresponding ASO. In a further feature, the sdRNA molecule can achieve gene regulation function not only in the cytoplasm of the cell, but also in the nucleus and / or mitochondria of the cell, and thus can target RNA in the nucleus and / or mitochondria. In a further feature, the sdRNA molecule silences the expression of the target gene in the cell more strongly or more effectively than a corresponding RNA duplex. Although the mechanism of action of the sdRNA of the present application is not clear, current studies show that the gene silencing potency of the sdRNA of the present application is not affected when Ago2 is knocked out, which indicates that the sdRNA of the present application can not act through an RISC-dependent mechanism. Thus, the sdRNA can be used to target a target gene or target a target sequence that is resistant or insensitive to siRNA.

[0042] In a second aspect, the present application provides a pharmaceutical composition comprising the composition of the first aspect as an active agent, and a pharmaceutically acceptable excipient, carrier or diluent thereof. Examples of such carriers include, but are not limited to: a pharmaceutical carrier, a positive charge carrier, a liposome, a lipid nanoparticle, 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 lipidoid.

[0043] In a third aspect, the present application provides a method of using the composition of the first aspect or the pharmaceutical composition of the second aspect to treat or prevent a disease or disorder, by administering a therapeutically effective amount of the sdRNA molecule of the present application or a pharmaceutical composition comprising the sdRNA molecule. The method of administration is selected from the following routes: intravenous (iv), subcutaneous (sc), oral (po), intramuscular (im), oral administration, inhalation, topical, intrathecal, and other site of administration.

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

[0045] In a fourth aspect, the present application provides a method of using the composition of the first aspect or the pharmaceutical composition of the second aspect to modulate or regulate gene expression or gene function in a eukaryotic cell. The method comprises the step of contacting the cell with an effective amount of any of the sdRNA molecules of the present application or a pharmaceutical composition comprising the sdRNA molecule.

[0046] In one embodiment, the contacting step comprises the step of introducing a composition comprising the sdRNA molecule into a target cell or organism in culture in which 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 lipidoid.

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

[0048] In one embodiment, the target gene is associated with a disease, pathological condition, or undesirable condition in a mammal. In a further embodiment, the target gene is a gene of a pathogenic microorganism. In a still further embodiment, the target gene is a viral gene. In another embodiment, the target gene is a tumor-associated gene. In 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 application provides an oligomeric duplex 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 of at least 8 nucleobases in length, wherein the ISD comprises a deoxyribonucleoside, an analog thereof, or a modified deoxyribonucleoside. The antisense sequence is at least 70% complementary to a target sequence.

[0050] Other features and advantages of the present application will be apparent from the additional description provided herein, including the different embodiments. The provided examples show different components and methods useful in practicing the present application. The examples do not limit the claimed application. Other components and methods useful in practicing the present application can be identified and employed by one skilled in the art as the present disclosure is read. Several embodiments have been shown and described, but any modifications are possible without departing from the spirit and scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

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

[0052] Figure 2A Exemplary structures of some embodiments of sdRNAs are shown, in which the antisense strand (first strand) of the sdRNA has at least one ISD and the sense strand (second strand) is pure RNA, as are exemplary sequences of sdRNAs targeting the APOCIII gene. In each duplex described herein, the sense strand is listed above the antisense strand.

[0053] Figure 2B Gene silencing efficacy of sdRNAs (structures and sequences as shown in Figure 2A ) is shown in comparison to the corresponding ASO (the corresponding ASO has the same antisense strand sequence as the sdRNA in Figure 2A ). Relative mRNA levels of the APOCIII gene were measured after transfection of 100 pM of the sdRNA and the corresponding ASO into HepaRG cells.

[0054] Figure 3A Exemplary structures of one embodiment of sdRNAs are shown, in which the antisense strand has at least one ISD and the sense strand is pure RNA, as are exemplary sequences of sdRNAs for targeting the APOB gene.

[0055] Figure 3B Gene silencing efficacy of sdRNAs designed to target the APOB gene (structures and sequences as shown in Figure 3A ) is shown. Relative mRNA levels of the APOB gene were measured after transfection of 5 nM of the sdRNA into HepaRG cells. DETAILED DESCRIPTION

[0056] The present invention relates to a novel gene or RNA modulation / silencing technology using short duplex RNA. This novel technology is used to modulate gene expression or function in vitro and in vivo by using short duplex RNA (sdRNA) compositions having deoxyribonucleotide spacers. The invention also provides methods for using the compositions to modulate the expression or function of target genes, or for treating or preventing diseases, as well as for other medical and biological applications. These compositions and methods provide high efficacy in modulating gene expression or gene function, and also reduce dose-dependent toxicity. 1. Definitions

[0057] As used herein, the singular form "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes multiple 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 those numbers. Generally, the term "about" is used herein to define the numerical value to a variation of 20%, 10%, 5%, or 1% above and below the stated value. In some embodiments, the term "about" is used to define the numerical value to a variation of 10% above and below the stated value. In some embodiments, the term "about" is used to define the numerical value to a variation of 5% above and below the stated value. In some embodiments, the term "about" is used to define the numerical value to a variation of 1% above and below the stated value.

[0059] As used herein, the terms "analog" or "analogue" are used interchangeably to mean functionally or structurally equivalent. For example, nucleoside and nucleotide analogs have been used for decades in the clinical treatment of cancer and viral infections, and researchers and the pharmaceutical industry are continually synthesizing and evaluating new compounds, see, e.g., Jordheim L.P. 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, analogs thereof, and modified deoxyribonucleosides. The term "deoxyribonucleotide monomer" refers to a nucleotide monomer including naturally occurring deoxyribonucleotides, analogs thereof, and modified deoxyribonucleotides.

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

[0062] As used herein, the term "nucleoside" refers to a compound comprising a nucleobase moiety and a sugar moiety. Nucleoside monomers include, but are not limited to, naturally occurring nucleosides (e.g., deoxyribonucleosides and ribonucleosides found in DNA and RNA, respectively), analogs thereof, and modified nucleosides. A nucleoside monomer can be a deoxyribonucleoside monomer or a ribonucleoside monomer. 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. Nucleotide 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. A nucleotide monomer can be a deoxyribonucleotide monomer or a ribonucleotide monomer. A modified nucleotide can be modified at one or more of the nitrogenous nucleobase moiety, the five-carbon sugar moiety, and the phosphate linking group that results in a change in the internucleoside linkage.

[0064] As used herein, the term "oligonucleotide" or "oligo" refers to a compound comprising a plurality of linked nucleoside monomers. In certain embodiments, one or more of the nucleoside monomers is modified, or one or more of the internucleoside linkages is 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)" refers to a molecule consisting of two strands of nucleotide monomers that hybridize to each other to form a duplex oligonucleotide and that is contacted with a cell or administered to a subject, wherein a 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 an antisense strand or a sense strand.

[0068] As used herein, the term "immediately adjacent" refers to the absence of intervening elements between two elements, e.g., 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, a modified internucleoside linkage, and / or a 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 phosphodiester linkage from 3' to 5'.

[0073] As used herein, the term "modified internucleoside linkage" refers to a substitution or any variation 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 naturally occurs in DNA (2-H) or RNA (2-OH).

[0075] As used herein, the term "modified sugar" refers to a substitution or variation from a natural sugar moiety. For example, a 2'-0-methoxyethyl modified sugar 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 furosyl sugar moiety includes a bridging group connecting two carbon atoms on the furosyl ring thereby forming a bicyclic sugar system.

[0078] As used herein, the term "2'-0-methoxyethyl" (also known as 2'-MOE, 2'-0(CH2)2— OCH3, and 2'-0-(2-methoxyethyl)) refers to a modification of the 2' position of a furosyl ring with an O-methoxyethyl group. A 2'-0-methoxyethyl modified sugar is a modified sugar. As used herein, the term "2'-0-methoxyethyl nucleotide" (also known as 2'-MOE RNA) refers to a modified nucleotide comprising a 2'-0-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. Conversely, "unmodified nucleobase" as used herein refers to adenine (A) and guanine (G) for purine bases, and thymine (T), cytosine (C), and uracil (U) for pyrimidine bases.

[0080] As used herein, the term "5-methylcytosine" refers to a 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 similarly to RNA. RNA-like nucleotides include, but are not limited to: bridged nucleic acid (BNA), LNA, cEt, 2'-0-methylated nucleotide, 2'-0-methoxyethylated (2'-MOE) nucleotide, 2'-fluorinated nucleotide, 2'-0- aminopropylated (2'-AP) nucleotide, tricyclo-DNA (tcDNA), and RNA surrogate.

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

[0083] As used herein, "non-coding RNA" refers to an RNA molecule that does not translate into a protein. Examples of non-coding RNA include transfer RNA (tRNA) and ribosomal RNA (rRNA), as well as small non-coding RNAs and long ncRNAs (IncRNAs). As used herein, examples of "small non-coding RNAs" include, but are not limited to: microRNAs (miRNAs), asRNAs, pre-miRNAs, pri-miRNAs, piRNAs, snoRNAs, snRNAs, exRNAs, scaRNAs, and any of the foregoing mimics. As used herein, "IncRNA," "long non-coding RNA" is a transcribed RNA molecule that contains more than 200 nucleotides that do not code for a protein. LncRNAs can also undergo common post-transcriptional modifications, including 5'-capping, 3'-polyadenylation, and splicing. Generally, IncRNAs are a diverse class of molecules that play a variety of roles in regulating the function of genes and genomes. For example, IncRNAs are known to regulate gene transcription, translation, and epigenetic regulation. Examples of IncRNAs include, but are not limited to: Kcnqlotl, Xlsirt, Xist, ANRIL, NEAT1, NRON, DANCR, OIP5-AS1, TUG1, CasC7, HOTAIR, and MALAT1. As used herein, "splice" or "splicing" refers to the natural process of removing unnecessary regions of RNA and reshaping the RNA. One example of modulating RNA target function by sdRNA is the modulation of non-coding RNA function. In some embodiments, the sdRNA is designed to target one of the foregoing small non-coding RNAs. In some embodiments, the sdRNA is designed to target a miRNA. In some embodiments, the sdRNA is designed to target a pre-miRNA. In some embodiments, the oligonucleotide or oligonucleotide duplex is designed to target a pri-miRNA. In some embodiments, the sdRNA is designed to target an IncRNA. In some embodiments, the sdRNA is designed to target a spliceosome.

[0084] Target RNA in the nucleus refers to an RNA molecule that is synthesized and / or functions in the nucleus of a cell. According to a preferred embodiment, the target RNA in the nucleus of the present application comprises non-coding RNA, IncRNA, pre-mRNA, and pre-miRNA. The term "pre-mRNA" as used herein refers to an unprocessed or partially processed mRNA precursor, containing introns and exons, synthesized by transcription from a cellular DNA template. The pre-mRNA needs to be spliced (introns removed) to produce an mRNA molecule containing only exons. In some embodiments, the sdRNA is designed to target a pre-mRNA. The terms "mitochondrial messenger RNA" and "mt-mRNA" refer to an mRNA molecule transcribed from mitochondrial DNA. In some embodiments, the sdRNA is designed to target a mt-mRNA in the mitochondria.

[0085] As used herein, the term "isolated" or "purified" refers to material that is substantially free of components that normally accompany the material in its natural 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 "spaced" refers to a section of a portion of different kinds, e.g., different kinds of nucleotides or nucleotide analogs, different modifications of the same kind of nucleotide or nucleotide analog, in adjacent space. In various embodiments of the present application, a "deoxynucleotide monomer interval segment (ISD)" refers to a section of deoxyribonucleotides in an oligonucleotide chain that has one or more deoxynucleotides and is connected to at least one portion of a different kind from the deoxynucleotide. For example: if the deoxynucleotide is unmodified, then the portion of a different kind can be a ribonucleotide or analog thereof, a modified ribonucleotide, a modified deoxynucleotide, or a deoxynucleotide analog. If the deoxynucleotide is modified, then the portion of a different kind can be a ribonucleotide or analog thereof, a modified ribonucleotide, an unmodified deoxynucleotide, a differently modified deoxynucleotide, or a different kind of deoxynucleotide analog.

[0087] As used herein, "modulate," "modulation," and grammatical equivalents thereof refer to an increase or decrease (e.g., silencing), in other words, upregulation or downregulation. 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 of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0088] As used herein, the terms "inhibit," "inhibiting," and grammatical equivalents when used in the context of a biological activity refer to a down-regulation of the biological activity, which can reduce or eliminate a target function (e.g., production of a protein, or phosphorylation of a molecule). In particular embodiments, inhibition can refer to a reduction of a 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 successful prevention of the onset of symptoms, reduction of symptoms, or elimination of the disease, condition (disease), or disorder.

[0089] As used herein, the term "substantially complementary" or "complementary" refers to complementarity in a double-stranded region between two strands having linked nucleosides, but not any single-stranded region (e.g., an overhanging end at the terminus or a gap region between two double-stranded regions). Complementarity need not be perfect; for example, there can be any number of base pair mismatches between the two strands having linked nucleosides. However, if the number of mismatches is so numerous that hybridization does not occur even under the most permissive hybridization conditions, then the sequence is not a substantially complementary sequence. In particular, when two sequences are referred to as "substantially complementary" in the present text, it is meant that the sequences are sufficiently complementary to each other that they can hybridize under selected reaction conditions. The relationship between nucleic acid complementarity and the stringency of hybridization is well known in the art. Two substantially complementary strands can be, for example, perfectly complementary, or can contain 1 to a number of mismatches, as long as hybridization conditions are sufficient to allow, for example, discrimination between a paired sequence and an unpaired sequence. Thus, a substantially complementary sequence can refer to a sequence 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 between any two of the aforementioned numbers, of base pair complementarity in a double-stranded region.

[0090] As used herein, "perfectly complementary" or "100% complementary" refers to each nucleobase in a sequence of nucleobases of a first strand having linked nucleosides having a complementary nucleobase in a second sequence of nucleobases of a second strand having linked nucleosides. In certain embodiments, the first strand having linked nucleosides is an antisense compound and the second strand having linked nucleosides is a target nucleic acid. In certain embodiments, the first strand having linked nucleosides is a sense compound and the second strand having linked nucleosides is an antisense compound, or vice versa.

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

[0092] The terms "administer," "administering," and "administration" are used herein in their broadest relative sense. These terms refer to any method of introducing a compound or pharmaceutical composition described herein to a subject, which can include, for example, introducing the compound systemically, locally, or in situ to the subject. Thus, the production of a compound disclosed herein in a subject by a composition, whether or not the composition includes the compound, is encompassed by these terms. When these terms are used in conjunction with "systemic" or "systemically," they generally refer to absorption of the compound or composition after it has been administered into the body, followed by distribution to 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 the intended result, including but not limited to disease treatment, as described below. In some embodiments, a "therapeutically effective amount" refers to an amount that is effective to detectably kill or inhibit 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, locally, 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 subject's body mass and age, the severity of the disease condition, the manner of administration, and the like), which can be readily determined by one of ordinary skill in the art. The term also applies to the dose that induces a particular response in target cells, for example, a reduction in cell migration. The specific dose can vary depending on, for example, the particular pharmaceutical composition, the subject and its age and existing health conditions or risks of health conditions, the dosage regimen to be followed, the severity of the disease, whether it is administered in combination with other agents, timing, the tissue to which it is administered, and the physical delivery system in which it is carried.

[0094] The term "cancer" in a subject refers to the presence of cells having the typical characteristics of cancerous cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain morphological features. Typically, cancerous cells will exist in the form of a tumor or mass, but cancerous cells can also exist alone in the subject, or can 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 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, adrenocorticoid carcinoma, uterine cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's Disease, esophageal cancer, gastroesophageal junction cancer, gastroesophageal adenocarcinoma, chondrosarcoma, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, Ewing's sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, testicular cancer, ureter cancer, renal pelvis cancer, mesothelioma, hepatocellular cancer, cholangiocarcinoma, renal cancer, renal cell carcinoma, chronic or acute leukemia, lymphocytic lymphoma, central nervous system (CNS) tumor, spinal axis tumor, brain stem glioma, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, medulloblastomas, meningiomas, squamous cell carcinoma, pituitary adenoma, refractory instances of any of the above cancers, or a combination of one or more of the above cancers. Some example cancers are included in general terms and are included in the term "cancer." For example, the general term urological cancer includes bladder cancer, prostate cancer, kidney cancer, testicular cancer, etc.; while another general term hepatobiliary system cancer includes liver cancer (which itself is a general term including hepatocellular carcinoma or cholangiocarcinoma), gall bladder cancer, cholangiocarcinoma, or pancreatic cancer. The present disclosure encompasses urological cancer and hepatobiliary system cancer and includes in the term "cancer."

[0095] The term "pharmaceutical composition" is a preparation containing a molecule or composition such as disclosed herein as an active ingredient, usually in admixture with other substances such as a pharmaceutical carrier, e.g., sterile water, to form a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or unit dosage form. Unit dosage forms are any of a variety of forms including, for example: a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial. The amount of active ingredient in unit dose of the composition is an effective amount and varies according to the particular therapeutic involved. Those skilled in the art will appreciate that dosages can need to be adjusted in accordance with the age and condition of the patient, and that the dosage will also depend on the route of administration. Multiple routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, and the like. Dosage forms for topical or transdermal administration of sdRNA molecules of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalers.

[0096] The term "agent" refers to a substance that can provide a therapeutic benefit when administered to an individual.

[0097] The term "pharmaceutically acceptable carrier" refers to a medium or diluent that does not interfere with the structure of the compound. Certain such carriers are capable of being formulated into pharmaceutical compositions such as tablets, pills, dragees, capsules, liquid formulations, gels, syrups, slurries, suspensions, and lozenges for oral ingestion by a subject. Certain such carriers are capable of being formulated into pharmaceutical compositions for injection, infusion, or topical administration. For example, the 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 variants, pharmaceutically acceptable salts, and other derivatives known in the art.

[0099] The term "pharmaceutically acceptable salt" refers to a salt of a compound that is physiologically and pharmaceutically acceptable, i.e., that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects thereto. The term "pharmaceutically acceptable salt" or "salt" includes salts of the parent compound prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic or organic acids and bases. The pharmaceutically acceptable salts of the compounds described herein can be prepared from the parent compound by well-known methods. For a review on 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 hydrochloride, hydrobromide, phosphate, sulphate, hydrogen sulphate, alkylsulphonate, arylsulphonate, acetate, benzoate, citrate, maleate, fumarate, succinate, lactate, and tartrate salts; 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 proven useful and are universally accepted for therapeutic administration to humans. Thus, in one embodiment, the compounds described herein are in the form of a sodium salt.

[0100] As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, who 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, the terms "treating", "treatment", "to treat", "alleviating" or "to alleviate" refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and / or halt the progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent or slow the development of a target pathologic condition or disorder. Thus those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. A subject is successfully "treated" according to the methods of the application if the subject exhibits one or more of the following: a reduction in the number of cancer cells or absence of cancer cells; a reduction in the tumor size; inhibition of cancer cell infiltration into peripheral organs including the spread of cancer into soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; and improved quality of life.

[0102] As used herein, the term "carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material involved in carrying or transporting the subject drug compound from one organ, or portion of the body, to another organ, or portion of the body. 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 carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate, magnesium stearate and polyethylene oxide-polypropylene oxide copolymer, 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 embodiments

[0103] In certain embodiments of the application, a duplex RNA composition is provided, wherein both the antisense strand and the sense strand are composed of linked nucleoside monomers. At least 50% or more of the nucleoside monomers in the entire duplex molecule are ribonucleoside monomers, some of which and / or the internucleoside linkages contained therein can be modified, i.e., modified from those found in natural RNA. The duplex RNA of the application further comprises one or more deoxynucleoside monomers in a deoxynucleotide interval segment (ISD). The one or more ISDs can be present in the antisense strand or the sense strand, or both. In some embodiments, each ISD independently consists of 1 deoxynucleotide monomer, or consists of 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous deoxynucleotide monomers. In some embodiments, the ISD has at least two contiguous and linked deoxynucleotide monomers.

[0104] The antisense strand and the sense strand of the short duplex RNA (sdRNA) molecules of the application are relatively short, wherein the length of the sense strand is at least equal to the length of the antisense strand, and thus are referred to as "short duplex RNA (sdRNA)". In addition, in certain embodiments, the two strands of the sdRNA molecules are of the same length, and the duplex molecules of the application can be more specifically referred to as "symmetric short duplex RNA".

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

[0106] In certain embodiments, the antisense strand and the sense strand of the same length form a symmetric structure without any overhang.

[0107] The compositions of the application can be used to modulate gene expression or function in eukaryotic cells in at least three ways: (i) contacting or administering to a subject a single sdRNA molecule; (ii) contacting or administering to a subject different kinds of sdRNA molecules at different times; (ii) contacting or administering to a subject different kinds of sdRNA molecules simultaneously.

[0108] In certain embodiments, an antisense strand includes a region of nucleobase sequences, 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 target gene, wherein the target gene includes an mRNA and a non-coding RNA. In certain embodiments, an antisense strand has a nucleobase sequence that includes a sequence that is fully complementary to a target segment of a targeted target gene. In certain embodiments, an antisense strand has a nucleobase sequence that, when hybridized to a target segment of a targeted target gene, includes no more than 1, 2, or 3 mismatches. In certain embodiments, a target gene is selected from an mRNA or a non-coding RNA associated with a mammalian disease. In certain embodiments, at least one ISD is distributed in the targeting region of an antisense strand. In certain embodiments, at least one ISD is located in a more central portion of an antisense strand (i.e., at least 1, 2, 3, 4, or 5 nucleobases from the ends of the strand, i.e., at position 2 or more central position from the end of the strand).

[0109] In various embodiments, an antisense strand has a backbone length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 linked nucleotide monomers, or an equivalent length, or a length range bracketed by any two of the aforementioned values (both end points of the range are included in the range). For example, some ranges of first antisense strand lengths include: 8-50 nucleotide monomers; 8-36 nucleotide monomers; 8-33 nucleotide monomers; 10-36 nucleotide monomers; 10-30 nucleotide monomers; 10-29 nucleotide monomers; 12-36 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; 14-24 nucleotide monomers; 15-23 nucleotide monomers; and 16-23 nucleotide monomers; and at least 8 nucleotide monomers.

[0110] In certain embodiments, the antisense strand is 10 to 36 (both of the range endpoints are included therein) nucleotide monomers in length. In other words, the antisense strand is 10 to 36 (both of the range endpoints are included therein) 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 of the range endpoints are included therein) linked nucleobases.

[0111] In certain embodiments, the antisense strand consists of 14 to 23 (both of the range endpoints are included therein) linked nucleotide monomers. In certain embodiments, the antisense strand consists of 20 linked nucleotide monomers. In certain embodiments, the antisense strand consists of 16 linked nucleotide monomers.

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

[0113] In one feature, the sense oligonucleotide strand is equal to or longer in length than the antisense oligonucleotide strand. In certain embodiments, the sense strand is from 1 to 16 linked nucleobases longer than the antisense strand full length. In certain embodiments, the sense strand is from 6 to 66 (both of the range endpoints are included therein) nucleotide monomers in length. In other words, these sense strands are from 6 to 66 (both of the range endpoints are included therein) linked nucleobase monomers in length. In other embodiments, the sense strand comprises an oligonucleotide consisting of from 20 to 21, 8 to 36, 10 to 30, 12 to 25, 14 to 24, or 16 to 23 (both of the range endpoints are included therein) linked nucleobases. In certain embodiments, the sense strand comprises an oligonucleotide, wherein the oligonucleotide consists of linked nucleobases having a 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, or 66, or a range defined by any two of the aforementioned numbers (both of the range endpoints are included therein). In some embodiments, the sense strand is a sense oligonucleotide.

[0114] In one feature, the length of the sense strand is equal to the length of the antisense strand. In certain embodiments, both ends of the double strand are blunt ends. In another feature, the sense strand has a backbone length that is at least the following number of nucleotide monomers longer than the antisense strand: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, and 20. In various embodiments, the sense 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, or 66 linked nucleotide monomers, or an equivalent length, or a length range bracketed by any two of the aforementioned values (both endpoints of the range are included). For example, in certain embodiments, ranges of sense strand lengths include: 6-66 nucleotide monomers; 8-50 nucleotide monomers; 8-40 nucleotide monomers; 8-36 nucleotide monomers; 8-33 nucleotide monomers; 10-36 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; 12-24 nucleotide monomers; 13-25 nucleotide monomers; 13-24 nucleotide monomers; 13-23 nucleotide monomers; 14-24 nucleotide monomers; 15-23 nucleotide monomers; 16-23 nucleotide monomers; and at least 8 nucleotide monomers. In certain embodiments, the sense strand can have a backbone length equal to or longer than any number of nucleotide monomers of the antisense strand, where the sense strand is capable of forming a thermodynamically stable duplex with the antisense strand.

[0115] In certain embodiments, the sense strand is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotide monomers longer than the antisense strand. In certain embodiments, the sense strand consists of 8-36 (both endpoints of the range are included) linked nucleotide monomers. In certain embodiments, the sense strand consists of 13 linked nucleoside monomers. In certain embodiments, the sense strand consists of 14 linked nucleoside monomers.

[0116] In certain embodiments of the application, the two strands of the sdRNA molecule form a symmetric duplex without any overhangs. In other various embodiments, the two ends of the second strand (the sense strand) are one of the following configurations: a 3' overhang and a 5' blunt end; a 5' overhang and a 3' blunt end; a 3' overhang and a 5' recessed end; a 3' recessed end and a 5' overhang; or a 3' overhang and a 5' overhang.

[0117] In certain embodiments, the 3' overhang of the sense 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 sense strand has a length of 1-26, 1-8, 14-26, 1-5, 1-3, or 1-2 nucleotide monomers (both end point values of the ranges are included therein).

[0118] In certain embodiments, the 5' overhang of the sense 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 sense strand has a length of 1-26, 1-8, 14-26, 1-5, 1-3, or 1-2 nucleotide monomers (both end point values of the ranges are included therein).

[0119] In the sdRNA molecules of the application, at least one of the nucleotide monomers in the first strand and / or the second strand 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 an aminophosphonate.

[0120] In some embodiments, the antisense and / or sense strands contain at least one modified internucleotide bond. This modified internucleotide bond may be between two ribonucleoside monomers, two deoxyribonucleoside monomers, or one deoxyribonucleoside monomer and one ribonucleoside monomer. Optionally, a phosphate group on at least one terminal nucleoside monomer may be modified. In some embodiments, the internucleotide bond is a thiophosphate internucleotide bond. In some embodiments, the internucleotide bond is a thioaminophosphate internucleotide bond. In some embodiments, each internucleotide bond in the oligonucleotide chain is a thiophosphate internucleotide bond. In some embodiments, all internucleotide bonds in the chain (antisense or sense strand or both) are thiophosphate internucleotide bonds, or a mixture of thiophosphate bonds and phosphodiester bonds.

[0121] In some embodiments, the antisense chain and / or sense chain comprises at least one nucleoside monomer having a modified sugar moiety. Such a nucleoside monomer may be a ribonucleoside monomer or a deoxyribonucleoside monomer.

[0122] In one embodiment, the 2' position of the modified sugar moiety is substituted with a group selected from the following: OR, R, halogen, SH, SR, NH2, NHR, NR2, or CN, wherein each R is independently a C1-C6 alkyl, alkenyl, or alkynyl group, and the 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 following: allyl, amino, azide, 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 Independently, it is H, or substituted or unsubstituted C1-C. 10alkyl. 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' (where R is H, C1-C 12 alkyl or protecting group), 4'-CH2—C(H)(CH3)-2', and 4'-CH2—C—(=CH2)-2'.

[0123] 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 (FANA), and methyl(methyloxy) (4'-CH(CH3)—O-2) bicyclic sugar (cEt).

[0124] In some embodiments, the antisense strand and / or the sense strand of a molecule of the application comprises at least one nucleotide monomer having a modified nucleobase. Such nucleoside monomers can be deoxyribonucleoside monomers or ribonucleoside monomers.

[0125] In some embodiments, the modified nucleobases are selected from the group consisting of 5-methylcytosine (5-Me-C), hypoxanthine, tritylated bases, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytidine, 1- methyl pseudouracil, 5-halouracil, and cytosine, 5-propynyl (-C≡C-CH3) uracil, and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo (particularly 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine, 2-F-adenine, 2- aminoadenine, 8-azaguanine and 8-azadenine, 7-deazaguanine and 7-deazaadenine, and 3- deazaguanine and 3-deazaadenine.

[0126] In particular embodiments, the modified nucleobases in the molecules of the application are 5-methylcytosine. In one embodiment, every cytosine base in the molecules of the application is 5-methylcytosine. In certain embodiments, the modified nucleobases are 5-methyluracil. In certain embodiments, every uracil is 5-methyluracil.

[0127] In certain embodiments, the antisense strand or the sense strand or both strands of the molecules of the application comprise linked ribonucleoside monomers and further comprise ISDs consisting of one or more linked deoxyribonucleoside monomers. In certain embodiments, the entire strand (either the antisense strand or the sense strand) consists of linked ribonucleoside monomers. In addition, there can be more ISD segments. The ISDs can be located anywhere in either strand. In certain embodiments, one or more ISDs are inserted into segments of ribonucleoside monomers, separating them into multiple segments. In certain embodiments, each ISD independently consists of 1 deoxynucleotide monomer, or consists of 2, 3, 4, 5, 6, 7, 8, 9, or 10 linked deoxynucleotide monomers.

[0128] In certain embodiments, at least half of the nucleotides in the sdRNA molecule are ribonucleotides. In various embodiments, at least 50%, 52%, 55%, 58%, 60%, 65%, or 70% of the nucleotides in the sdRNA molecule are ribonucleotide monomers.

[0129] In certain embodiments, the total number of deoxynucleotide monomers in an sdRNA molecule is no more than the total number of ribonucleotide monomers in the same sdRNA molecule. Another feature is that the total number of deoxynucleotide monomers in an ISD of an sdRNA molecule is at least 10%, 15%, 20%, or 25% of the total number of nucleotide monomers in the sdRNA. In certain embodiments, the total number of deoxynucleotide monomers in either strand of an sdRNA molecule is no more than the total number of ribonucleotide monomers in the same strand of the sdRNA molecule. In certain embodiments, the total number of deoxynucleotide monomers in the first strand of an sdRNA molecule is no more than the total number of ribonucleotide monomers in the same first strand of the sdRNA molecule. The total number of deoxynucleotide monomers in the second strand of an sdRNA molecule is no more than the total number of ribonucleotide monomers in the same second strand of the sdRNA molecule. In certain embodiments, the total number of deoxynucleotide monomers in an sdRNA molecule is no more than the total number of ribonucleotide monomers in the same sdRNA molecule, and the total number of deoxynucleotide monomers in one of the strands of the sdRNA molecule can be more than the total number of ribonucleotide monomers in the same strand of the sdRNA molecule.

[0130] In certain embodiments, at least one or each linked deoxynucleotide monomer of an ISD is a modified deoxynucleotide or a deoxynucleotide analog. The deoxynucleotide can be modified in the same or similar manner as follows: with a modified internucleoside linkage, a modified sugar moiety, and / or a modified nucleobase.

[0131] 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).

[0132] In some embodiments, the sugar moiety of the ribonucleotide monomer is selected from the group consisting of: a naturally occurring ribonucleotide (2-OH), a 2'-F modified sugar, a 2'-OMe modified sugar, a 2'-O-methoxyethyl modified sugar (MOE), a 4'-(CH2)— O-2' bicyclic sugar (LNA), and a methyl(alkylenoxy)(4'-CH(CH3)— O-2) bicyclic sugar (cEt).

[0133] In certain embodiments, at least one or each deoxynucleoside monomer in the antisense strand, the sense strand, or both strands has a sugar moiety modified with 2'-deoxy-2'-fluoroarabinose (FANA). In one embodiment, the ISD comprises at least one nucleoside monomer having a sugar moiety modified with FANA. In another embodiment, all of the nucleoside monomers in the ISD have a sugar moiety modified with FANA. In another embodiment, all of the nucleosides in the ISD are naturally occurring deoxynucleosides. In one embodiment, all of the nucleosides in the ISD are naturally occurring deoxyribonucleosides, or have a sugar moiety modified with FANA.

[0134] In certain embodiments, at least one or each ribonucleoside monomer in the antisense strand, the sense strand, or both strands has a sugar moiety selected from the group consisting of 2'-0-methoxyethyl modified sugar (MOE), 4'-(CH2)-0-2' bicyclic sugar (LNA), and methyl(methyloxy) (4'-CH(CH3)-0-2) bicyclic sugar (cEt) modified sugar moieties.

[0135] In certain embodiments, each internucleoside linkage between the deoxynucleoside monomers of each ISD is a phosphorothioate linkage. In certain embodiments, each internucleoside linkage between the deoxynucleotide monomers of each ISD is a natural phosphate linkage without phosphorothioate modification.

[0136] In certain embodiments, each deoxynucleoside monomer in each ISD has a FANA modification, and wherein each cytosine is a 5-methylcytosine. In certain embodiments, each deoxyribonucleoside monomer in each ISD has a FANA modification, wherein each cytosine is a 5-methylcytosine, and each internucleoside linkage is a phosphorothioate linkage.

[0137] In certain embodiments, the molecules of the application have an antisense strand or a sense strand composed of ribonucleoside monomers, wherein each internucleoside linkage is a phosphorothioate linkage. In certain embodiments, the molecules of the application have an antisense strand or a sense strand composed of ribonucleoside monomers, wherein each internucleoside linkage is a natural phosphate linkage without phosphorothioate modification.

[0138] In certain embodiments, the molecules of the application include a sense strand, wherein the sense strand includes the same modifications as the complementary antisense strand segment.

[0139] Exemplary structures of sdRNA molecules of the application are shown in Figure 2A and 3A .

[0140] In certain embodiments, the short symmetrical duplex RNA and at least one ISD in the antisense strand of the duplex molecule are capable of achieving robust gene silencing. The data shown in all the examples below demonstrate that the short symmetrical duplex RNA based on the novel platform technology of the present application, i.e., antisense oligoribonucleotide with at least one ISD, can achieve extremely robust gene silencing. Our data on the gene silencing activity of sdRNA and the SAR factor suggest that the gene silencing profile of sdRNA is quite different from that of siRNA and ASO, which suggests a novel and unique mechanism of gene silencing that remains to be elucidated.

[0141] In certain embodiments, the molecules of the present application can be stabilized against degradation by at least one chemical modification or secondary structure. The sense oligonucleotide strand and the antisense oligonucleotide strand can have unpaired or imperfectly paired nucleotide monomers. The sense oligonucleotide strand and / or the antisense oligonucleotide strand can have one or more nicks (a cut in the nucleic acid backbone), gaps (a fragment strand with one or more missing nucleotides), and modified nucleotides or nucleotide analogs. Not only can any or all of the nucleotide monomers in the sense oligonucleotide strand and the antisense oligonucleotide strand be chemically modified, but each strand can be conjugated to one or more moieties or ligands to enhance its functionality, e.g., with a moiety or ligand selected from the group consisting of a polypeptide, an antibody, an antibody fragment, a polymer, a polysaccharide, a lipid, a hydrophobic moiety or molecule, a cationic moiety or molecule, a lipophilic compound or moiety, an oligonucleotide, a cholesterol, a GalNAc, and an aptamer.

[0142] In certain embodiments, the duplex molecule of the present application does not contain any mismatches or bulges in the duplex region, and the two strands are perfectly complementary to each other in the duplex region. In another embodiment, the duplex contains mismatches and / or bulges in the duplex region.

[0143] 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 IncRNA. In certain embodiments, the target is a mt-mRNA. As long as the antisense strand is substantially complementary to the target sequence, the antisense strand occupies the target by hybridizing to the target sequence, inactivating the target gene. 3. Unpaired or mismatched regions

[0144] The region of complementarity between the antisense strand and the sense strand of the sdRNA of the present application can have at least one unpaired or imperfectly paired region, e.g., one or more mismatches. Mismatches in the sense strand are sometimes needed to reduce off-target effects or to achieve other functionalities of the sdRNA.

[0145] As is well known to those skilled in the art, mismatched bases can be introduced without abolishing activity. Similarly, the antisense strand of the sdRNA of the application can include regions that are not paired or mismatched when base pairing with the targeted RNA. Mismatches in the antisense strand are sometimes required to reduce off-target effects or to achieve other functions of the sdRNA. 4. Modifications

[0146] A nucleoside monomer is a base-sugar combination. The nucleobase (also referred to as a base) portion of a nucleoside monomer is typically a heterocyclic base moiety. A nucleotide monomer is a nucleoside monomer further including a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleoside monomers that include a pentofuranosyl sugar, the phosphate group can be linked to the 2’, 3’ or 5’ hydroxyl moiety of the sugar. An oligonucleotide is formed by covalently linking together adjacent nucleoside monomers to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are often referred to as forming inter-nucleoside linkages.

[0147] Modifications to the sdRNA molecules, antisense strands, and / or sense strands of the application include substitutions or alterations to the inter-nucleoside linkages, sugar moieties, or nucleobases. Modified sdRNAs, antisense strands, and / or sense strands are in some instances preferred over their native forms for desirable characteristics, such as increased inhibitory activity, enhanced cellular uptake, increased strand affinity, solubility, reduced non-specific interactions, and resistance to RNase degradation or enhanced stability. Thus, results similar to those obtained with short antisense strands having such chemically modified nucleoside monomers can generally be obtained. One or more of the natural nucleotides in the antisense strands and sense strands of the application can be replaced by modified nucleotides or nucleotide analogs. The substitutions can occur at any position in the antisense strands and sense strands.

[0148] Modifications to oligonucleotide molecules have been investigated to improve the stability of various oligonucleotide molecules, including antisense oligonucleotides, ribozymes, aptamers, and RNAi (Chiu and Rana, 2003; Czauderna et al., 2003; de Fougerolles et al., 2007; Kim and Rossi, 2007; Mack, 2007; Zhang et al., 2006; Schrnidt, 2007; Setten RL et al., 2020; Crooke ST et al., 2018; and Roberts TC et al., 2020).

[0149] Any stabilizing modification known to those skilled in the art can be used to improve the stability of the oligonucleotide molecule. Within the oligonucleotide molecule, chemical modifications can be introduced into the phosphate backbone (e.g., phosphorothioate linkages), sugar (e.g., locked nucleic acids, glycerol nucleic acids, cEt, 2’-MOE, 2’-fluoro uridine, 2’-O-methyl), and / or base (e.g., 2’-fluoro pyrimidines).

[0150] The following sections summarize several examples of such chemical modifications.

[0151] In various embodiments, the modified nucleotide or nucleotide analog is a sugar-modified, backbone-modified, and / or base-modified nucleotide. 4.1 Modified internucleoside linkage or backbone-modified nucleotide

[0152] The naturally occurring internucleoside linkage in RNA and DNA is a 3’ to 5’ phosphodiester linkage. The sdRNA molecules of the application having one or more modified internucleoside linkages (i.e., non-naturally occurring internucleoside linkages) in one strand or in both strands are sometimes selected for having desirable properties (e.g., enhanced cellular uptake, enhanced affinity for a target nucleic acid, and increased stability in the presence of nucleases) compared to the corresponding molecule having only naturally occurring internucleoside linkages.

[0153] Oligonucleotide strands having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom and internucleoside linkages that do not have a phosphorus atom. In one embodiment, the phosphodiester internucleoside linkage can be modified to include at least one of a nitrogen heteroatom or a sulfur heteroatom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, thiophosphoramidate, and phosphorothioate. Methods of making phosphorus-containing and non-phosphorus-containing linkages are well known.

[0154] In one embodiment, the modified nucleotide or nucleotide analog is a backbone-modified nucleotide. Backbone-modified nucleotides can have a modification on the phosphodiester internucleoside linkage. In another embodiment, the backbone-modified nucleotide is a phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage. 4.2 Modified sugar moiety

[0155] The antisense strands and / or sense strands of the application can optionally contain one or more nucleoside monomers with modified sugar moieties. These sugar-modified nucleoside monomers can confer enhanced nuclease stability, increased binding affinity, or some other advantageous biological property to the antisense strands and / or sense strands. In certain embodiments, the nucleoside monomers comprise chemically-modified furanose ring moieties. Examples of chemically-modified furanose rings include, but are not limited to: addition of substituents; including 5' and 2' substituents, non-geminal ring atom bridging to form bicyclic nucleic acids (BNAs), replacement of the ribosyl ring oxygen atom with S, N(R) or C(R1)(R2) (R, R1and R2are each independently H, C1-C6alkyl or a protecting group), and combinations thereof. Examples of chemically-modified sugars include 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO 2008 / 101157 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 US 2005-0130923 published June 16, 2005) or alternative 5'-substitution for BNAs (see PCT International Application WO 2007 / 134181 published November 22, 2007, wherein the LNA is substituted, e.g., 5'-methyl or 5'-vinyl). 12 The antisense strands and / or sense strands of the application can optionally contain one or more nucleoside monomers with modified sugar moieties. These sugar-modified nucleoside monomers can confer enhanced nuclease stability, increased binding affinity, or some other advantageous biological property to the antisense strands and / or sense strands. In certain embodiments, the nucleoside monomers comprise chemically-modified furanose ring moieties. Examples of chemically-modified furanose rings include, but are not limited to: addition of substituents; including 5' and 2' substituents, non-geminal ring atom bridging to form bicyclic nucleic acids (BNAs), replacement of the ribosyl ring oxygen atom with S, N(R) or C(R1)(R2) (R, R1and R2are each independently H, C1-C6alkyl or a protecting group), and combinations thereof. Examples of chemically-modified sugars include 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO 2008 / 101157 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 US 2005-0130923 published June 16, 2005) or alternative 5'-substitution for BNAs (see PCT International Application WO 2007 / 134181 published November 22, 2007, wherein the LNA is substituted, e.g., 5'-methyl or 5'-vinyl).

[0156] Examples of nucleoside monomers with 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)2OCH3substituents. The substituents at the 2' position can also be selected from the group consisting of allyl, amino, azido, thio, O-allyl, O-C1-C6alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-O-N(R1)2, O-CH2-C(=O)-N(R1)2, and O-CH2-C(=O)-N(R1)-(CH2)2-N(R1)2, wherein each R1is independently H, or substituted or unsubstituted C1-C6alkyl. 10 m n m n m n m n 10 Examples of nucleoside monomers with 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)2OCH3substituents. The substituents at the 2' position can also be selected from the group consisting of allyl, amino, azido, thio, O-allyl, O-C1-C6alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-O-N(R1)2, O-CH2-C(=O)-N(R1)2, and O-CH2-C(=O)-N(R1)-(CH2)2-N(R1)2, wherein each R1is independently H, or substituted or unsubstituted C1-C6alkyl.

[0157] ​​​​​​​​​Bicyclic nucleosides are modified nucleosides having a bicyclic sugar moiety. Examples of bicyclic nucleic acids (BNA) include, without limitation, nucleosides comprising a bridging group between the 4' and 2' ribosyl ring atoms. In certain embodiments, the sdRNAs, antisense strands, and / or sense strands provided herein include one or more BNA nucleosides, the bridging group in the BNA nucleosides 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 7,399,845, issued July 15, 2008); 4'-C(CH3)(CH3)—O-2' (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 US 2004-0171570 published on September 2, 2004); 4'-CH2—N(R)—O-2', wherein R is H, C1-C6alkyl, or a protecting group (see U.S. Patent 7,427,672, issued September 23, 2008); 4'-CH2—C(H)(CH3)-2' (see Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2—C—(=CH2)-2' (and analogs thereof, see PCT / US2008 / 066154 published as W2008 / 154401 on December 8, 2008). 12 alkyl or a protecting group (see U.S. Patent 7,427,672, issued September 23, 2008); 4'-CH2—C(H)(CH3)-2' (see Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2—C—(=CH2)-2' (and analogs thereof, see PCT / US2008 / 066154 published as W2008 / 154401 on December 8, 2008).

[0158] In certain embodiments, bicyclic nucleosides include, but are not limited to: (A) a-L-methyleneoxy (4'-CH2— O-2) BNAs, (B) β-D-methyleneoxy (4'-CH2— O-2) BNAs, (C) ethyleneoxy (4'-(CH2)2— O-2') BNAs, (D) aminooxy (4'-CH2— O— N(R)-2') BNAs, (E) oxymo amino (4'-CH2— N(R)— O-2) BNAs, (F) methyl(methyleneoxy) (4'-CH(CH3)— O-2) BNAs (also known as constrained ethyl or cEt), (G) methylenethio (4'-CH2— S-2') BNAs, (H) methyleneamino (4'-CH2— N(R)-2') BNAs, (I) methylcarbocyclic (4'-CH2— CH(CH3)-2) BNAs, (J) propylene carbocyclic (4'-(CH2)3-2') BNAs, and (K) vinyl BNAs.

[0159] In certain embodiments, the modified nucleotide or nucleotide analog is a sugar-modified ribonucleotide in which the 2'-OH group is replaced by a group selected from 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 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 a 2'-OMe modified nucleotide, a 2'-F modified nucleotide, a 2'-O-methoxyethyl (2' MOE) modified nucleotide, a LNA (locked nucleic acid) modified nucleotide, a GNA (glycerol nucleic acid) modified nucleotide, and a cEt (constrained ethyl) modified nucleotide. In one embodiment, the sugar-modified deoxynucleotide is a FANA modified deoxynucleotide.

[0160] Chemical modifications at the 2' position of the ribose sugar can stabilize the molecules of the application, for example, 2'-O-methyl purines and 2'-fluoro pyrimidines can increase their resistance to endonuclease activity in serum. The site of introduction of the modification should be carefully selected to avoid significantly decreasing the ability of the molecule to silence / modulate. In certain embodiments, the first nucleotide monomer adjacent to the 5'-terminal nucleotide monomer of the antisense strand is a 2'-fluoro ribonucleotide. 4.3 Modified nucleobases

[0161] The anti-sense and / or sense strand of the sdRNA molecule can also have modified or substituted nucleobases (or bases). Nucleobase (or base) modifications or substitutions are structurally non-natural occurring or synthetic unmodified nucleobases, but are functionally interchangeable therewith. Both natural and modified nucleobases are capable of participating in hydrogen bonding. The nucleobase modifications can confer nuclease stability, binding affinity or some other advantageous biological property to the sdRNA molecule. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-Me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of the anti-sense and sense strands. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

[0162] 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, 2-thiacytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3) uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo (particularly 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

[0163] Heterocyclic base moieties can include those in which the purine or pyrimidine base is substituted with other heterocycles, such as 7-deazadenine, 7-deazaguanine, 2- aminopyridine, and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of the anti-sense 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.

[0164] 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.

[0165] Any modified nucleotide or analog that can be advantageous for stability or affinity can be made without departing from the spirit and scope of the application. Several examples of such chemical modifications are the same as summarized above. 5. Pharmaceutical compositions

[0166] In some embodiments, the present application also provides a pharmaceutical formulation comprising an sdRNA of the present application or a pharmaceutically acceptable derivative thereof and at least one pharmaceutically acceptable excipient or carrier. As used herein, "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in "Remington: The Science and Practice of Pharmacy, Twentieth Edition," Lippincott Williams & Wilkins, Philadelphia, PA, incorporated herein by reference. Examples of such carriers or diluents include, but are not limited to: water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers, such as fixed oils can also be used. Such materials are well known in the art and are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA. Such media and agents are capable of being used in conjunction with the sdRNA molecules without causing a significant degradation of the molecule. Except insofar as any conventional media or agent is incompatible with the sdRNA molecule, such as by producing an incompatibilized compound, use thereof in the compositions is contemplated.

[0167] Examples of pharmaceutically acceptable carriers that can be used with the molecules of the present application include, but are not limited to: pharmaceutical carriers, positive charge 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.

[0168] In certain embodiments, the present application provides a method of treatment comprising administering to a subject in need thereof 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 (po), intramuscular (im) injection, oral administration, inhalation, topical, intrathecal, and other modes of administration. In another embodiment, the therapeutically effective amount is from 1 ng to 1 g per day, from 100 ng to 1 g per day, or from 1 μg to 1000 mg per day.

[0169] Formulation methods are disclosed in PCT International Application PCT / US02 / 24262 (WO 03 / 011224), U.S. Patent Application Publication No. 2003 / 0091639, and U.S. Patent Application Publication No. 2004 / 0071775, each of which is incorporated herein by reference.

[0170] The sdRNA molecules of the present application are administered in a suitable dosage form prepared by combining a therapeutically effective amount (e.g., an effective level sufficient to achieve the desired therapeutic effect by inhibiting tumor growth, killing tumor cells, treating or preventing a cell proliferative disorder, etc.) of the sdRNA molecules of the present application (as the active ingredient) with standard pharmaceutical carriers or diluents in accordance with conventional procedures (i.e., producing pharmaceutical compositions of the present application).

[0171] These steps can involve the appropriate mixing, granulating, and compressing or dissolving of the ingredients, as required, to obtain the desired formulation. In another embodiment, a therapeutically effective amount of the sdRNA molecules are administered in a suitable dosage form without standard pharmaceutical carriers or diluents. In some embodiments, a therapeutically effective amount of the duplex molecules of the present application are administered in a suitable dosage form. Pharmaceutically acceptable carriers include solid carriers such as lactose, calcium phosphate, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, and the like. Exemplary liquid carriers include syrup, peanut oil, olive oil, water, and the like. Similarly, the carrier or diluent can include time delay material known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax, ethyl cellulose, hydroxypropylmethyl cellulose, methymethacrylate, and the like. Other

[0172] The pharmaceutical compositions of the present application can be prepared in a manner widely understood in the art, e.g., by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping or lyophilizing processes. One or more physiologically acceptable carriers can be used in the formulation of the pharmaceutical compositions in a conventional manner, including excipients and / or auxiliaries which facilitate processing of the sense and antisense oligonucleotides into a pharmaceutically acceptable formulation. The appropriate formulation depends on the route of administration chosen.

[0173] The compositions, compounds, combinations or pharmaceutical compositions of the present application can be administered to a subject in a number of well-known ways for chemotherapy treatment. For example, for the treatment of cancer, the sdRNA molecules of the present application can be injected directly into a tumor, injected into the bloodstream or body cavity, or administered orally or via a skin patch. For the treatment of psoriatic conditions, either systemic administration (e.g., oral administration) or local administration to the affected skin area are preferred routes of administration. The dosage chosen should be sufficient to constitute effective treatment, but not so high as to cause unacceptable side effects. During treatment and for a reasonable period of time after treatment, the condition of the disease (e.g., cancer, psoriasis, etc.) and the health of the patient should be closely monitored. 6. Uses 6.1 Methods of use

[0174] The present application also provides a method of modulating 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 sdRNA molecule as disclosed herein under conditions in which selective gene silencing can occur, and the sdRNA molecule can mediate the production of selective gene silencing against a target nucleic acid having a sequence portion that is substantially complementary to the antisense strand of the sdRNA molecule. The target nucleic acid can be an RNA, such as an mRNA, pre-mRNA, mt-mRNA, or non-coding RNA, wherein these RNAs either encode a protein associated with a disease or modulate a biological pathway associated with a disease.

[0175] In one embodiment, the contacting step comprises introducing the sdRNA molecule into a target cell or organism in culture under conditions in which selective gene silencing can occur. In another embodiment, the introducing step comprises mixing, transfection, lipofection, infection, electroporation, or other delivery technique. In another embodiment, the introducing step comprises administering intravenously, subcutaneously, intrathecally, orally, by inhalation, topically, or other clinically acceptable method of administration using a pharmaceutically acceptable excipient, carrier, or diluent selected from the group consisting of a pharmaceutical carrier, a positive charge 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 lipidoid.

[0176] In one embodiment, the silencing method is used to determine the function or utility of a gene in a cell or organism.

[0177] In one embodiment, the gene or RNA targeted by the compositions of the application is associated with a disease, such as a human disease or an animal disease, a pathological condition or an undesirable 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 derived from a virus. In another embodiment, the target gene or target RNA is associated with a tumor.

[0178] In an alternative embodiment, the gene or RNA targeted by the compositions of the application is a gene or RNA associated with a cancer, an autoimmune disease, an inflammatory disease, a degenerative disease, an infectious disease, a proliferative disease, a metabolic disease, an immune-mediated disorder, an allergic disease, a dermatological disease, a malignancy, a gastrointestinal disease, a liver disease, a respiratory disorder, a cardiovascular disorder, a dermatological disease, a renal disease, a rheumatoid disease, a neurological disorder, a psychiatric disorder, an endocrine disorder, or a disease or disorder associated with aging. 6.2 Methods of treatment

[0179] The present application also provides methods of treating or preventing various diseases or conditions, including those summarized for ASOs and siRNAs (Czech, 2006; de Fougerolles et al., 2007; Dykxhoorn et al., 2003; Kim and Rossi, 2007; Mack, 2007; Crooke ST et al., 2018; Setten RL et al., 2019; Roberts TC et al., 2020). The methods comprise administering to a subject in need an effective amount of sdRNA molecules under conditions in which the desired genetic inhibition (described in Section 6.1 above) can occur.

[0180] In an exemplary embodiment, a therapeutically effective amount of a pharmaceutical composition is administered to a subject in need to treat or prevent a disease or undesirable condition, wherein the pharmaceutical composition has sdRNA molecules and a pharmaceutically acceptable excipient, carrier or diluent.

[0181] In some embodiments, the present application can be used for cancer treatment or prevention of cancer. The sdRNA compositions can be used to silence or knock down genes associated with cell proliferation disorders or malignancies. Examples of such genes are k-Ras, beta-catenin, Stat3. These oncogenes are active in and associated with a large number of human cancers.

[0182] The novel compositions of this invention can also be used to treat or prevent eye 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 arthritis, autoimmune diseases), and skin diseases.

[0183] In another embodiment, the method of administration is selected from the following routes: intravenous injection (iv), subcutaneous injection (sc), oral administration (po), intrathecal, inhalation, local and regional application. Example

[0184] The embodiments provided below further illustrate different features of the invention. The embodiments also illustrate useful methods for carrying out the invention. These examples do not limit the claimed invention. Methods and Materials Cell Culture

[0185] HepaRG cells were grown in William medium supplemented with 10% FBS, 10 mg / ml hydrocortisone, and 4 mg / ml recombinant human insulin. Other suitable commercially available cell lines known to those skilled in the art are available for purchase and use. sdRNA transfection of cells

[0186] 24 hours before transfection, seed HepaRG cells or other commonly used cell lines into 6-well plates (1x10⁻⁶ cells per well). 5 (cells / 2mL / well). Following the preparation method described, through... RNAiMAX (Thermo Fisher, USA) was used to transfect sdRNA. In short, sdRNA and RNAiMAX were incubated in serum-free OPTI-MEM (Thermo Fisher) for 20 minutes and then added to cells containing culture medium. Quantitative PCR

[0187] Transfected cells were harvested 48 hours after transfection with the specified sdRNA. RNA was isolated using TRIZOL and qRT-PCR was performed using TaqMan one-step RT-PCR reagent. The APOCIII assay was used to detect APOCIII mRNA; the APOB assay was used to detect APOB mRNA, etc. The GAPDH mRNA level was used as an internal control.

[0188] target sequence

[0189] To verify the gene silencing effect of the sdRNA disclosed in this invention, sdRNAs targeting different genes were designed and manufactured. Figure 1 Exemplary target genes, target sequences, and corresponding antisense strand sequences of sdRNAs designed and used in the following embodiments are shown. Example 1: Structure-activity relationship (SAR) study of sdRNAs with ISD distribution in AS with PS modification

[0190] Figure 2A An exemplary structure and sequence of an sdRNA implementation targeting the APOCIII gene is shown, wherein ISD is present on the antisense strand (AS) and the sense strand (SS) consists entirely of linked ribonucleotide monomers. Figure 2A The antisense oligonucleotide (ASO) with the same structure and sequence as the antisense strand of sdRNA1 was also designed as the corresponding ASO for comparison. The gene silencing effects of these structures were tested. Specifically, the gene silencing activity of sdRNA1 and the corresponding ASO at 100 pM was tested in HepaRG cells.

[0191] exist Figure 2A In the diagram, all letters "D" in the shown structure represent DNA residues or deoxyribonucleotide monomers; all letters "R" in the shown structure represent RNA residues or ribonucleotide monomers, including 2'-MOE-modified RNA residues or 2'-MOE-modified ribonucleotide monomers; all letters "rR" in the shown structure represent RNA residues or ribonucleotide monomers, including naturally occurring RNA residues or ribonucleotide monomers; all "*" in the shown structure represent PS (phosphothiophosphate nucleoside internucleotide bonds). All lowercase letters "a, c, g, t" in the sequence represent DNA residues; all uppercase letters "A, C, G, U" in the sequence represent 2'-MOE-modified RNA residues; and all underlined uppercase letters "... A , C , G , U " represents RNA residues, where all "U"s are 5-methyluridine 2'-MOE RNA residues; where all "U"s are RNA residues. U" are 5-methylcytosine RNA residues; wherein all "C" are 5-methylcytosine 2'-MOE RNA residues; wherein all "c" are 5-methylcytosine DNA residues; wherein all "G" are guanosine RNA residues; and wherein all "g" are guanosine DNA residues. C " are 5-methylcytosine RNA residues; all "*" in the sequence represent PS (phosphorothioate internucleoside linkage).

[0192] Figure 2B The results in Table 1 show that sdRNA1 (structure and sequence as shown in Figure 2A ) has very strong gene silencing activity at very low concentration (picomolar level) and is stronger and more potent than the corresponding single-stranded ASO (i.e. ISIS 304801) that is optimized with the most advanced technology. Example 2: Structure-activity relationship (SAR) study of sdRNAs with ISD distribution in AS without modification of the internucleoside linkage

[0193] Figure 3A An exemplary structure of one embodiment of sdRNA targeting APOB gene is shown, wherein ISD is distributed in AS, and each internucleoside linkage between adjacent nucleoside monomers of the sdRNA molecule is a naturally occurring internucleoside linkage (i.e. phosphodiester linkage) (labeled as sdRNA2 in Figure 3A ). The gene silencing effect of this sdRNA targeting APOB gene was tested. Specifically, the gene silencing activity of sdRNA2 at 5 nM was tested in HepaRG cells.

[0194] In Figure 3A , all letters "D", "R" and "rR" in the illustrated structure, and lower case letters "a, c, g, t", capital letters "A, C, G, U", underlined capital letters A , C , G , U " in the illustrated sequence have the same meaning as indicated in Figure 2A .

[0195] Figure 3B It is shown that sdRNA2 (structure and sequence as shown in Figure 3A ) exhibits strong gene silencing activity against the intended target APOB gene at low concentration in HepaRG cells.

[0196] The results in Example 1-2 strongly suggest that sdRNAs designed according to the principles of the present application can achieve very strong gene silencing potency against different example genes.

[0197] In other examples of detecting the gene silencing effect of sdRNAs disclosed herein, sdRNAs were designed to target pre-mRNAs in the nucleus, IncRNAs in the nucleus and mt-mRNAs in mitochondria, and the same method as described in the above examples was used for detection, and the results of quantitative PCR showed that sdRNAs designed according to the principles of the present application could achieve very strong gene silencing efficacy, while siRNAs designed to target the same RNAs in the nucleus and mitochondria could not show gene silencing activity. EQUIVALENTS

[0198] The representative examples are intended to help illustrate the application, and are not intended to, nor should they be construed to, limit the scope of the application. Indeed, various modifications of the application and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples and the references to the scientific and patent literature included herein. The examples contain important additional information, exemplification and guidance to adapt the certain embodiments of the application to various situations as well as equivalents of the application.

[0199] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can 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 otherwise specified in the particular sequences disclosed. INcorporation BY REFERENCE

[0200] Other documents have been cited and referenced in the present disclosure, such as patents, patent applications, patent publications, journals, books, papers, web contents. All such documents are hereby incorporated by reference in their entirety for all purposes. Any material, or portion thereof, that is said to be incorporated by reference into this document, but which contradicts any definition, statement, or other disclosure actually in this document, is only incorporated into this document to the extent that the incorporating citation is consistent with this document. REFERENCES 1. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21 -nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001 May 24; 411(6836):494-8. doi: 10.1038 / 35078107. PMID: 11373684. 2. Sun Xiangao, Rogoff Harry A, Li Chiang J. Asymmetric RNA duplexes mediate RNA interference in mammalian cells. Nat Biotechnol. 2008 Dec; 26(12): 1379-82. doi: 10.1038 / nbt.1512. Epub 2008 Nov 23. Erratum in: Nat Biotechnol. 2009 Feb; 27(2):205. PMID: 19029911. 3. C. Frank Bennett and Eric E. Swayze, RNA Targeting Therapeutics: Molecular Mechanisms of Antisense Oligonucleotides as a Therapeutic Platform. Annu. Rev. Pharmacol. Toxicol. 2010. 50:259-93. 4. C. Frank Bennett. Therapeutic Antisense Oligonucleotides Are Coming of Age. Annu Rev Med. 2019 Jan 27; 70:307-321. doi: 10.1146 / annurev-med-041217-010829. PMID: 30691367. 5. Setten RL, Rossi JJ, Han SP. The current state and future directions of RNAi-based therapeutics. Nat Rev Drug Discov. 2019 Jun; 18(6): 421-446. doi: 10.1038 / s41573-019-0017-4. Erratum in: Nat Rev Drug Discov. 2019 Mar 18;: Erratum in: Nat Rev Drug Discov. 2019 Apr 24;: PMID: 30846871. 6. Sibley CR, Seow Y, Wood MJ. Novel RNA-based strategies for therapeutic gene silencing. Mol Ther. 2010 Mar; 18(3): 466-76. doi: 10.1038 / mt.2009.306. Epub 2010 Jan 19. PMID: 20087319; PMCID: PMC2839433. 7. Grimm D. Asymmetry in siRNA design. Gene Ther. 2009 Jul; 16(7): 827-9. doi: 10.1038 / gt.2009.45. Epub 2009 Apr 30. PMID: 19404320. 8. Crooke ST, Witztum JL, Bennett CF, Baker BF. RNA-Targeted Therapeutics. Cell Metab. 2018 Apr 3; 27(4): 714-739. doi: 10.1016 / j.cmet.2018.03.004. Erratum in: Cell Metab. 2019 Feb 5; 29(2): 501. PMID: 29617640. 9. Roberts TC, Langer R, Wood MJA. Advances in oligonucleotide drug delivery. Nat Rev Drug Discov. 2020 Oct; 19(10): 673-694. doi: 10.1038 / s41573-020-0075-7. Epub 2020 Aug 11. PMID: 32782413; PMCID: PMC7419031. 10. Ryszard Kole, Adrian R. Krainer, Sidney Altman, RNA therapeutics: Beyond RNA interference and antisense oligonucleotids. Nat Rev Drug Discov. 2016. 11(2): 125-140. 11. CyA. Stein, Daniela Castanotto, FDA-Approved Oligonucleotide Therapies in 2017. Molecular Therapy. 2017. Vol. 25 No 5 May 2017 12. Richard G. Lee, Jeff Crosby, Brenda F. Baker, Mark J. Graham, Rosanne M. Crooke, Antisense Technology: An Emerging Platform for Cardiovascular Disease Therapeutics. J. of Cardiovasc. Trans. Res. 2013. DOI 10.1007 / s12265-013-9495-7 13. Zamecnik, P. C., & Stephenson, M. L. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proceedings of the National Academy of Sciences USA 75, 1978. 280-284. 14. Stanley T. Crooke, Molecular Mechanisms of Antisense Oligonucleotides. NUCLEIC ACID THERAPEUTICS. Volume 27, Number 2, 2017 MaryAnn Liebert, Inc. DOI: 10.1089 / nat.2016.0656 15. Antisense Drug Technologies: Principles, Strategies, and Applications. 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 and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998. 391, 806-811 17. de Fougerolles A, Vornlocher HP, Maraganore J, Lieberman J. Interfering with disease: a progress report on siRNA-based therapeutics. Nature Rev Drug Discov. 2007; 6:443-453. [PubMed: 17541417] 18. Jackson AL, Bartz SR, Schelter JM, Kobayashi SV, Burchard J, et al. 2003. Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21:635-37 19. Lin X, Ruan X, Anderson MG, McDowell JA, Kroeger P, et al. 2005. siRNA-mediated off-target gene silencing triggered by a 7nt complementation. Nucleic Acids Res. 33:4527-35 20. Kwoh JT. 2008. An overview of the clinical safety experience of first- and second-generation antisense oligonucleotides. See Ref. 9, pp. 365-99 21. Henry SP, Kim T-W, Kramer-Strickland K, Zanardi TA, Fey RA, Levin AA. 2008. Toxicological properties of 2'-O-methoxyethyl chimeric antisense inhibitors in animals and man. See Ref. 9, pp. 327-63 22. Geary, RS.; Yu, RZ.; Levin, AA. Antisense Drug Technologies: Principles, Strategies, and Applications. See Ref. 9, pp. 183-217 23. 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Claims

1. A short double-stranded RNA (sdRNA) molecule comprising a first strand and a second strand, wherein the first strand and the second strand each comprise linked nucleotide monomers, 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; wherein the second strand is equal to or longer in length than the first strand; wherein, comprises at least one deoxynucleotide monomer interval segment (ISD) in the first strand or the second strand or both strands of the sdRNA molecule, the ISD comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deoxynucleotide monomers; and wherein the total number of deoxynucleotide monomers in the sdRNA molecule is not more than the total number of ribonucleotide monomers in the sdRNA molecule.

2. The sdRNA molecule of claim 1, the sdRNA with the ISD has at least one improved genetic regulatory or pharmaceutical property compared to the corresponding short double-stranded RNA without the ISD.

3. The sdRNA molecule of claim 2, wherein the at least one improved genetic regulatory or pharmaceutical property comprises: (a) triggering gene silencing at picomolar concentrations, such as at 500 pM, 300 pM, 200 pM, 100 pM or lower concentrations; (b) achieving gene silencing in the cytoplasm as well as in the nucleus and mitochondria; (c) eliminating or reducing interference with endogenous microRNA function; (d) allowing more extensive chemical modifications, including non-RNA-like nucleotide modifications or substitutions, or (e) reducing synthesis cost or increasing stability.

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

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

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

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

8. The sdRNA molecule of any one of claims 1-7, wherein the at least one ISD is distributed in the at least one targeting region of the first strand and the at least one double-stranded region of the second strand.

9. The sdRNA molecule of any one of claims 1 to 8, wherein the at least one ISD comprises at least 4 contiguous deoxynucleotide monomers.

10. The sdRNA molecule of claim 1, wherein the first strand is at least 70%, 80%, 85%, 90%, 95% complementary or fully complementary to the target segment of the target RNA.

11. The sdRNA molecule of claim 1, wherein the second strand is at least 70%, 75%, 80%, 85%, 90%, 95% complementary or fully complementary to the first strand.

12. The sdRNA molecule of claim 1, wherein the length of the first strand is selected from the group consisting 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, and 50 nucleotide monomers.

13. The sdRNA molecule of claim 1, wherein the length of the first strand is selected from the group consisting of: a) 6-50 nucleotide monomers, b) 8-40 nucleotide monomers, c) 8-36 nucleotide monomers, d) 8-33 nucleotide monomers, e) 10-30 nucleotide monomers, and f) 8-29 nucleotide monomers.

14. The sdRNA molecule of claim 1, wherein the length of the second strand is equal to the length of the first strand.

15. The sdRNA molecule of claim 1, wherein the second strand is at least selected from the group consisting of the following number of nucleotide monomers longer than the first strand: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

16. The sdRNA molecule of claim 1, wherein the length of the second strand is selected from the group consisting of: 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, and 50 nucleotide monomers.

17. The sdRNA molecule of claim 1, wherein the length of the second strand is selected from the group consisting of: a) 6-50 nucleotide monomers, b) 8-40 nucleotide monomers, c) 8-36 nucleotide monomers, d) 8-33 nucleotide monomers, e) 8-32 nucleotide monomers, f) 8-30 nucleotide monomers, g) 8-29 nucleotide monomers, and h) 8-25 nucleotide monomers.

18. The sdRNA molecule of claim 1, wherein the double-stranded region consists of base pairs selected from the group consisting of the following numbers of base pairs: 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 base pairs.

19. The sdRNA molecule of claim 1, wherein (a) both ends of the sdRNA double strand are blunt ends; (b) both the first strand and the second strand have 3 '-overhangs; or (c) both the first strand and the second strand have 5 '-overhangs.

20. The sdRNA molecule of claim 15, wherein the second strand has one of the following configurations: 3' overhang and 5' overhang, 3' overhang and 5' blunt end, 3' blunt end and 5' overhang, 3' overhang and 5' recessed end, or 5' overhang and 3' recessed end.

21. The sdRNA molecule of claim 1, wherein the first strand has one of the following configurations: 3' overhang and 5' blunt end, 3' blunt end and 5' overhang, 3' overhang and 5' recessed end, 5' overhang and 3' recessed end, 3' blunt end and 5' blunt end, or 3' recessed end and 5' recessed end.

22. The sdRNA molecule of claim 20 or 21, wherein the 3' overhang or the 5' overhang has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide monomers.

23. The sdRNA molecule of any one of claims 1-22, wherein at least one nucleotide monomer is a modified nucleotide or a nucleotide analog.

24. The sdRNA molecule of claim 23, wherein the modified nucleotide or nucleotide analog is a sugar-modified, backbone-modified, and / or base-modified nucleotide.

25. The sdRNA molecule of claim 24, wherein the backbone-modified nucleotide has a modification on the internucleoside linkage.

26. The sdRNA molecule of claim 25, wherein the internucleoside linkage is modified to comprise at least one of a nitrogen heteroatom or a sulfur heteroatom.

27. The sdRNA molecule of claim 26, wherein the modified internucleoside linkage is selected from the group consisting of: a phosphorothioate group (P=S), a phosphotriester, a methylphosphonate, and a phosphoramidate.

28. The sdRNA molecule of claim 23, wherein (a) 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; or (b) the ISD region of the second strand comprises at least one modified internucleoside linkage, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

29. The sdRNA molecule of claim 28, wherein (a) each internucleoside linkage of the first strand is a phosphorothioate internucleoside linkage; and / or (b) each internucleoside linkage of the second strand is a phosphorothioate internucleoside linkage.

30. The sdRNA molecule of claim 23, 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 a C1-C6 alkyl, alkenyl, or alkynyl, and halogen is F, Cl, Br, or I. (b) The 2' position of the modified sugar moiety is replaced by a group selected from the group consisting of: allyl, amino, azide, thio, O-allyl, O-Cl-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(R 1) -(CH2)2-N(R m (R) n ), where each R1, R m and R n Independently, it is 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 replaced with a bicyclic sugar selected from the group consisting of: 4'-(CH2)-0-2' (LNA), 4'-(CH2)-S-2', 4'-(CH2)2-0-2' (ENA), 4'-CH(CH3)-0-2' (cEt), and 4'-CH(CH2OCH3)-0-2', 4'-C(CH3)(CH3)-0-2', 4'-CH2-N(OCH3)-2', 4'-CH2-0-N(CH3)-2', 4'-CH2-N(R)-0-2' (where 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'-0- methoxyethyl modified sugar (MOE), 4'-(CH2)— O-2' bicyclic sugar (LNA), 2'-deoxy-2'-fluoroarabino (FANA), and methyl (methylenoxy) (4'-CH(CH3)-O-2) bicyclic sugar (cEt).

31. The sdRNA molecule of any one of claims 1-22, 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'-fluoroarabino (FANA).

32. The sdRNA molecule of claim 23, 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), inosine nucleobase, 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, 1-methyl-pseudouracil, 5-halouracil and cytosine, 5-propynyl (-CºC-CH3) uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azadenine, 7-deazaguanine and 7-deazadenine, and 3-deazaguanine and 3-deazadenine. (b) the modified nucleobase is 5-methylcytosine; and / or (c) each cytosine base is 5-methylcytosine.

33. The sdRNA molecule of any one of claims 1-32, wherein the sdRNA has the ability to modulate gene expression or function in a cell, wherein the cell is a eukaryotic cell, wherein the eukaryotic cell is a mammalian cell.

34. The sdRNA molecule of claim 1, wherein the target RNA is an mRNA, pre-mRNA, mt-RNA, or a non-coding RNA, wherein these RNAs encode a protein associated with a disease or disorder or modulate a biological pathway associated with a disease or disorder.

35. The sdRNA molecule of claim 1, wherein the target RNA is selected from the group consisting of: a) an mRNA, pre-mRNA, or mt-RNA of a gene associated with a disease or disorder in humans or animals, b) an mRNA or pre-mRNA of a gene of a pathogenic microorganism, c) a viral RNA d) a IncRNA, e) a miRNA, and f) an RNA associated with a disease selected from the group consisting of an autoimmune disease, an inflammatory disease, a degenerative disease, an infectious disease, a proliferative disease, a metabolic disease, an immune-mediated disorder, an allergic disease, a skin disease, a malignant disease, a gastrointestinal disease, a respiratory disorder, a cardiovascular disorder, a kidney disease, a rheumatic disease, a neurological disorder, an endocrine disorder, and a disease associated with aging.

36. The sdRNA molecule of any one of claims 1-35, wherein the first strand and / or the second strand is conjugated to a ligand or moiety.

37. The sdRNA molecule of claim 36, 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, a cholesterol, a GalNAc, and an aptamer.

38. A pharmaceutical composition comprising as an active agent the sdRNA molecule of any one of claims 1-37 and a pharmaceutically acceptable excipient, carrier, or diluent.

39. The pharmaceutical composition of claim 38, wherein the carrier is selected from the group consisting of a drug carrier, a positive charge carrier, a lipid nanoparticle, a liposome, a protein carrier, a hydrophobic moiety or molecule, a cationic moiety or molecule, a GalNAc, a polysaccharide polymer, a nanoparticle, a nanoemulsion, a cholesterol, a lipid, a lipophilic compound or moiety, and a lipidoid.

40. A method of treating or preventing a disease or disorder, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of the sdRNA molecule of any one of claims 1-37 or the pharmaceutical composition of claim 38 or 39.

41. The method of claim 40, wherein the disease or disorder is selected from the group consisting of a cancer, an autoimmune disease, an inflammatory disease, a degenerative disease, an infectious disease, a proliferative disease, a metabolic disease, an immune-mediated disorder, an allergic disease, a skin disease, a malignant disease, a gastrointestinal disease, a liver disease, a respiratory disorder, a cardiovascular disorder, a skin disease, a kidney disease, a rheumatic disease, a neurological disorder, a psychiatric disorder, an endocrine disorder, and a disorder or disease associated with aging.

42. The method of claim 41, wherein the sdRNA molecule or pharmaceutical composition is administered by a route selected from the group consisting of intravenous (iv), subcutaneous (sc), oral (po), intramuscular (im), oral administration, inhalation, topical, intrathecal, and administration at other sites.

43. A method of modulating gene expression or gene function in a eukaryotic cell, wherein the method comprises contacting the cell with an effective amount of a sdRNA molecule of any one of claims 1-37 or a pharmaceutical composition described in claim 38 or 39.

44. A short double-stranded RNA (sdRNA) molecule comprising a first strand and a second strand, wherein both the first strand and the second strand comprise linked nucleotide monomers, wherein the nucleotide monomers are selected from the group consisting of nucleotides, analogs thereof, and modified nucleotides, wherein the length of the first strand is equal to the length of the second strand, or the length of the first strand is shorter than the length of the second strand by a number of monomers selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8 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 836 (both end point values of the range are included therein) nucleoside monomers linked by bonds, wherein the bonds are selected from the group consisting of phosphorothioate bonds, phosphodiester bonds, and 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 10-36 (both end point values of the range are included therein) nucleoside monomers linked 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 sdRNA molecule comprises at least one deoxyribonucleotide monomer spacer (ISD) linked to at least one ribonucleotide monomer, wherein the ribonucleotide monomer is selected from the group consisting of ribonucleotides, analogs thereof, and modified ribonucleotides, wherein the ISD in the sdRNA molecule comprises at least one deoxynucleotide monomer, wherein the deoxynucleotide monomer is selected from the group consisting of deoxynucleotides, analogs thereof, and modified deoxynucleotides, and wherein the total number of deoxynucleotide monomers in the sdRNA molecule does not exceed the total number of ribonucleotide monomers in the sdRNA molecule.

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