Short double-stranded DNA as a novel gene silencing technology and its uses

Abstract

The present invention discloses a novel type of gene silencing technology for modulating target nucleic acid and / or protein in cells, tissues, organisms and animals. This new technology provides a composition for use in gene silencing applications, including the prevention and treatment of human disease. The composition comprises a short, double-stranded DNA molecule, in which the sense strand is at least equal in length to the antisense strand. The double-stranded DNA molecule further comprises at least one interspersed ribonucleotide monomer. The present invention further provides a method of using the composition to modulate the expression or function of a target gene, or for the treatment or prevention of disease, as well as for other medical or biological applications.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of co-pending U.S. Provisional Patent Application No. 63 / 195,010, filed May 29, 2021, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention relates to short double-stranded DNA used as a gene silencing technology, and to related compositions and methods that can be used in biological or medical research, in the treatment and prevention of diseases, and for applications of gene silencing in other fields of biology. [Background technology]

[0003] 2. Background of the Invention Modern medical therapeutics rely on two fundamental technologies: small molecule chemistry and protein / antibody technology. However, only about 10% of targets identified by genomic and biomedical research can be addressed by the aforementioned two fundamental technologies. Oligonucleotides have the potential to address a vast number of targets, including those that are not druggable by small molecule chemistry and antibody / protein technology. More than 40 years of research have produced antisense oligonucleotide (ASO) and small interfering RNA (siRNA) technologies (Cy A. Stein et al., 2017). However, despite more than 40 years of research, significant druggability issues have prevented the development of ASO and siRNA technologies from becoming mainstay therapeutic platforms, except for a few clinical orphan indications. Such druggability issues include low silencing efficiency, off-target effects, stimulation of unintended immune responses, tissue penetration challenges, and in vivo delivery, among others. Thus, there is a significant unmet need to generate novel technologies to target genes of interest in various biological and medical applications.

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

[0005] RNAi is a mechanism by which short double-stranded RNA induces the loss of homologous RNA, first observed in plants and demonstrated in Caenorhabditis elegans (A. Fire et al., 1998). The mechanism involves the degradation of long dsRNA into short interfering double-stranded RNA (siRNA) and the interaction of siRNA with the multiprotein RNA-induced silencing complex (RISC), where the siRNA unwinds, the sense strand is discarded, and the antisense or guide strand binds to the RISC endonuclease AGO2, which then cleaves the target RNA (de Fougerolles et al., 2007; Ryszard Kole, 2016). In mammalian cells, synthetic siRNAs or asymmetric short interfering RNAs (aiRNAs or asymmetric siRNAs) can be used to induce gene silencing through a RISC-dependent mechanism (see Elbashir SM et al., 2001; Sun X et al., 2008; U.S. Pat. Nos. 7,056,704 and 9,328,345).

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

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

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

[0009] [Patent Document 1] U.S. Patent No. 7,919,472 [Patent Document 2] U.S. Patent No. 9,045,754 [Patent Document 3] U.S. Patent No. 7,056,704 [Patent Document 4] U.S. Patent No. 9,328,345 Summary of the Invention [Means for solving the problem]

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

[0011] In the present invention, short double-stranded DNA (sdDNA) molecules are further interspersed with one or a small number of ribonucleotide monomers, which form at least one ribonucleotide monomer interspersed segment ("ISR").

[0012] The high gene silencing effect of the novel platform technology based on sdDNA contained in the present disclosure is achieved in one embodiment by the sense strand of the oligonucleotide monomer and the antisense strand of the oligonucleotide monomer that is substantially complementary to the target ribonucleotide sequence. The data of the present inventors show that the sdDNA molecules of the present invention can induce gene silencing at picomolar concentrations due to their unique and novel composition, which is more potent than ASO, siRNA and other existing gene silencing technologies, and thus allows for reduced dose-dependent toxicity. The sdDNA molecules of the present invention are also expected to have at least one of the following advantages over existing gene silencing technologies, including better tissue penetration; enabling gene silencing in the nucleus, mitochondria, etc., in contrast to siRNA-based gene silencing that only occurs in the cytoplasm; reduced off-target effects; better stability; elimination or reduction of undesired competition with endogenous microRNA pathways associated with siRNA; lower synthesis costs and other improved pharmaceutical properties. Therefore, the sdDNA molecules of the present invention have great potential to address various challenges faced by existing technologies. The sdDNA molecules of the present invention can be used in any area where current oligonucleotides are applied or intended to be used, including research, diagnosis, disease prevention and treatment, and other applications in the biological fields, including agricultural and veterinary medicine.

[0013] In a first aspect, the present invention provides a composition comprising a short double-stranded DNA (sdDNA) molecule having a first strand and a second strand. The length of the second strand is at least equal to the length of the first strand, more specifically, the second strand is longer than or equal to the length of the first strand. The first strand is substantially complementary to a segment targeted by the at least one targeting region of the RNA targeted, and therefore may be considered to be an antisense strand or an antisense oligonucleotide. Furthermore, the second strand, which may be considered to be a sense strand or a sense oligonucleotide, is substantially complementary to the first strand and forms at least one double-stranded region with the first strand. The sdDNA molecule comprises at least one ribonucleotide monomer interspersed segment (ISR). In one aspect, the ISR in the sdDNA molecule comprises at least one ribonucleotide monomer that may be present in either or both strands.

[0014] The compositions provided by the present invention are used to modulate gene expression or function in eukaryotic cells, and sdDNA is contacted with the cells or administered to a subject.

[0015] In some embodiments, the sdDNA molecule comprises at least one or at least two ribonucleotide monomer interspersed segments (ISRs). In a feature of the sdDNA molecule of the present invention, the first strand of the molecule may comprise at least one ISR, or the second strand of the molecule may comprise at least one ISR. In an embodiment, the first strand comprises at least one ISR, and the second strand also comprises at least one ISR. In a feature, each ISR, independently of the other, consists of one ribonucleotide monomer or comprises at least two, three, four, five, or six consecutive ribonucleotide monomers. In another feature, the ISR comprises at least two ribonucleotide monomers, whether they are consecutive or separated by at least one (one, two, three, four, five, six, seven, eight, nine, ten, or more) intervening monomer of a different type. In yet another feature, the total number of ribonucleotide monomers of all ISRs contained within the first strand is at least two.

[0016] In one feature, at least one ISR is located in at least one targeting region of the first (antisense) strand. In another feature, at least one ISR is located in at least one double-stranded region of the second (sense) strand. In yet another feature, at least one ISR is located in at least one targeting region of the first strand, and at least one ISR is located in at least one double-stranded region of the second (sense) strand. In some embodiments, at least one ISR can be located anywhere in the first strand. In some embodiments, at least one ISR is located at or near the 5' end of the first strand (within 7 nucleobases or within 33% of the total number of nucleobases in the strand, counting from the end to the end, e.g., position numbers 1, 2, 3, 4, 5, 6 or 7 for a strand that is about 21 nucleobases long, starting from the end); and / or at or near the 3' end of the first strand (within 7 nucleobases or within 33% of the total number of nucleobases in the strand, counting from the end to the end); and / or more centrally in the first strand. In some embodiments, the at least one ISR located in the first strand is located only in the overhang region of the first strand. In some embodiments, the at least one ISR located in the first strand is located in both the overhang region and the double-stranded region of the first strand. In some embodiments, the ISR in the first strand comprises at least one ribonucleotide monomer located at the 5' end of the first strand or the 3' end of the first strand. In some embodiments, at least one ISR is located at or near the 5'-end of the second strand (within 7 nucleobases, or within 33% of the total number of nucleobases in said strand, counting from the end to the end); and / or at or near the 3'-end of the second strand (within 7 nucleobases, or within 33% of the total number of nucleobases in said strand, counting from the end to the end); and / or more centrally in the second strand.

[0017] In one feature, the first strand or antisense strand comprises a plurality of linked nucleotide monomers forming a nucleobase sequence, and is at least 70%, 80%, 85%, 90%, 95% or fully complementary to the targeted segment of the RNA of the targeted gene. In certain embodiments, the targeted RNA is selected from an mRNA or a non-coding RNA, which encodes a protein involved in a disease, such as a mammalian disease, or controls a part of a biological pathway involved in said disease. The terms "target" and "targeted" are used interchangeably in this disclosure and share the same meaning.

[0018] In various embodiments, the first strand (the antisense strand) has a backbone length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 linked nucleotide monomers, or an equivalent thereof, or a range bounded by any two of the foregoing values ​​(including both endpoints of the range). For example, some of the ranges of lengths of the first strand include: (a) 8 to 36 nucleotide monomers; (b) 8 to 33 nucleotide monomers; (c) 10 to 30 nucleotide monomers; (d) 10 to 29 nucleotide monomers; (e) 12 to 29 nucleotide monomers; (f) 12 to 28 nucleotide monomers; (g) 12 to 26 nucleotide monomers; (h) 12 to 25 nucleotide monomers; (i) 13 to 25 nucleotide monomers; (j) 13 to 24 nucleotide monomers; (k) 13 to 23 nucleotide monomers; (l) 14 to 24 nucleotide monomers; (m) 15 to 23 nucleotide monomers; and (n) 8 to 50 nucleotide monomers; (o) 16 to 23 nucleotide monomers; (p) 10 to 36 nucleotide monomers; and (p) at least 8 nucleotide monomers.

[0019] In one aspect, the second or sense strand comprises a plurality of linked nucleotide monomers forming a nucleobase sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% complementary or fully complementary to the linked region of the first or antisense strand, such that the two strands form a double-stranded region that comprises 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 fully complementary to at least one linked region of the first / antisense strand, and forms at least one double-stranded region without any mismatch.In some embodiments, the sense strand is complementary to at least one linked region of the first / antisense strand, and forms at least one double-stranded region with one, two, three or more mismatches.In one feature, the mismatch monomer in the sense strand has a nucleobase selected from the group consisting of A, G, C and T, or a modified nucleobase.

[0020] In one feature, the second strand or sense strand has the same backbone length as the first strand or antisense strand. In an embodiment, the two strands of the sdDNA molecule form a symmetric duplex without any overhang. In another embodiment, both the first strand and the second strand have a 3'-overhang or a 5'-overhang. In an embodiment, the 3'-overhang and the 5'-overhang of the two strands have the same length of at least 1, 2, 3, 4 or 5 nucleotide monomers.

[0021] In other features, the second or sense strand has a backbone length that is longer than the first or antisense strand by, or at least, the following number of nucleotide monomers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, the 3'-overhang of the second strand has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotide monomers, or a range between any two of the preceding values ​​(including both endpoints of the range). In certain embodiments, the 5'-overhang of the second strand has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotide monomers, or a range between any two of the preceding values ​​(including both endpoints of the range). In certain embodiments of the invention, the second strand has a 3'-overhang of 1-5 nucleotide monomers, and a 5'-overhang of 1-5 nucleotide monomers (including both endpoints of the range). In another embodiment, the second strand has a 3'-overhang of 1-8 nucleotide monomers (including both endpoints of the range), and a 5' blunt end. In yet another embodiment, the second strand has a 5'-overhang of 1-8 nucleotide monomers (including both endpoints of the range), and a 3' blunt end. In yet another embodiment, the second strand has a 3'-overhang of 1-10 nucleotide monomers (inclusive of both endpoints of the range) and a 5' recessed end, or a 5'-overhang of 1-10 nucleotide monomers (inclusive of both endpoints of the range) and a 3' recessed end.

[0022] In various embodiments, the second or sense 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 thereof, or a range bounded by any two of the foregoing values ​​(including both endpoints of the range). In certain embodiments, for example, a portion of the range of lengths of the second sense strand includes: (a) 8 to 36 nucleotide monomers; (b) 8 to 33 nucleotide monomers; (c) 10 to 30 nucleotide monomers; (d) 10 to 29 nucleotide monomers; (e) 12 to 29 nucleotide monomers; (f) 12 to 28 nucleotide monomers; (g) 12 to 26 nucleotide monomers; (h) 12 to 25 nucleotide monomers; (i) 12 to 24 nucleotide monomers; (j) 13 to 25 nucleotide monomers; (k) 13 to 24 nucleotide monomers; (l) 13 to 23 nucleotide monomers; (m) 14 to 24 nucleotide monomers; (n) 15 to 23 nucleotide monomers; (o) 8 to 50 nucleotide monomers; (p) tides of 16 to 23 nucleotide monomers; (q) 10 to 36 nucleotide monomers; and (r) at least 8 nucleotide monomers.

[0023] In certain aspects of the sdDNA molecules of the present invention, at least one nucleotide monomer in the first and / or second strand is a modified nucleotide or nucleotide analog, such as a sugar-modified nucleotide, a backbone-modified nucleotide, and / or a base-modified nucleotide. In certain embodiments, such backbone-modified nucleotides have at least a modification of the internucleoside linkage, for example, to include at least one of a nitrogen or sulfur heteroatom. In some embodiments, the modified internucleoside linkage is or includes a phosphorothioate (P=S) group, a phosphotriester, a methylphosphonate, or a phosphoramidate.

[0024] In certain embodiments, the first and / or second strands comprise at least one modified internucleoside linkage, and the modified internucleoside linkage is a phosphorothioate internucleoside linkage. In some embodiments, at least one internucleoside linkage between deoxyribonucleotide monomers of the second strand is a phosphorothioate internucleoside linkage. In some embodiments, each internucleoside linkage of the first and / or second strand is a phosphorothioate internucleoside linkage. In various embodiments, the internucleoside linkage is a mixture of phosphorothioate and phosphodiester linkages of the first and / or second strands.

[0025] In one aspect, the first and / or second strand of the molecules of the invention comprise at least one modified nucleotide or nucleotide analog comprising a modified sugar moiety. In certain embodiments, the 2' position of the modified sugar moiety is replaced with a group selected from OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, where each R is independently C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I. In some embodiments, the 2' position of the modified sugar moiety is replaced with a group selected from allyl, amino, azido, thio, O-allyl, O-C1-C6 alkyl, alkenyl, or alkynyl. 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 R l , R m and R n are independently H or substituted or unsubstituted C1-C 10In some embodiments, the modified sugar moiety has a substituent selected from the group of 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, 2'-OCH2CH3, 2'-OCH2CH2F, 2'-O-aminopropylated (2'-AP), and 2'-O(CH2)2OCH3. In some embodiments, the modified sugar moiety is substituted with a bicyclic sugar selected from the group of 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (cEt), and 4'-CH(CHOCH3)-O-2', 4'-C(CH3)(CH3)-O-2', 4'-CH2-N(OCH3)-2', 4'-CH2-ON(CH3)-2', 4'-CH2-N(R)-O-2' (wherein R is H, C1-C12 alkyl, or a protecting group), 4'-CH2-C(H)(CH3)-2', and 4'-CH2-C-(=CH2)-2'. In some embodiments, the modified sugar moiety is selected from the group of 2'-O-methoxyethyl modified sugars (MOE), 4'-(CH2)-O-2' bicyclic sugars (LNA), 2'-deoxy-2'-fluoroarabinose (2'-F-arabino, FANA), and methyl(methyleneoxy) (4'-CH(CH3)-O-2) bicyclic sugars (cEt).

[0026] In one aspect of the sdDNA molecules of the invention, the sugar moieties of the deoxyribonucleotide monomers are either sugar moieties of naturally occurring deoxyribonucleotides (2-H) or 2'-deoxy-2'-fluoroarabinose (FA).

[0027] In one aspect of the sdDNA molecules of the invention, the sugar moieties of the ribonucleotide monomers are selected from naturally occurring ribonucleotides (2-OH), 2'-F modified sugars, 2'-OMe modified sugars, 2'-O-methoxyethyl modified sugars (MOE), 4'-(CH2)-O-2' bicyclic sugars (LNA), and methyl(methyleneoxy) (4'-CH(CH3)-O-2) bicyclic sugars (cEt).

[0028] In another aspect, the first and / or second strand of the molecules of the invention comprise at least one nucleotide monomer that comprises a modified nucleobase. In some embodiments, the modified nucleobase is 5-methylcytosine (5-Me-C), inosine bases, tritylated bases, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine; 5-propynyl (-C≡C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases; 6-azouracil, cytosine and thymine, 5-uracil, cytosine, thymine, cytosine ... The modified nucleic acid base is selected from the group consisting of 5-methyluridine, 4-methyluridine, 1-methyl-pseudouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-methyluridine and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, and 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. In certain embodiments, the modified nucleic acid base is 5-methylcytosine. In certain embodiments, each cytosine base in the molecules of the invention is 5-methylcytosine. In certain embodiments, each uridine base in the ISR of the sdDNA molecules of the invention is 5-methyluridine.

[0029] In one aspect, an sdDNA molecule of the invention can contain at least one CpG motif that can be recognized by a pattern recognition receptor (PRR), such as a Toll-like receptor.

[0030] In one aspect, the first and / or second strands of the molecules of the invention are conjugated to a ligand or moiety. In certain embodiments, the ligand or moiety is selected from the group of peptides / proteins, antibodies, polymers, polysaccharides, lipids, hydrophobic moieties or molecules, cationic moieties or molecules, lipophilic compounds or moieties, oligonucleotides, cholesterol, GalNAc, and aptamers.

[0031] In one aspect of the invention, sdDNA molecules are used to modulate gene expression or function in cells, eg, eukaryotic cells, such as mammalian cells.

[0032] In certain embodiments, the targeted RNA, which directs at least a portion of the nucleotide monomer sequence of the sdDNA molecule according to the principles of the present invention, is selected from mRNA or non-coding RNA, which encodes a protein involved in a disease or controls a portion of a biological pathway involved in a disease. Such targeted RNA may, in various embodiments, be selected from mRNA of a gene involved in a human or animal disease or condition; mRNA of a gene of a pathogenic microorganism; viral RNA, and RNA involved in a disease or disorder selected from the group consisting of autoimmune diseases, inflammatory diseases, degenerative diseases, infectious diseases, proliferative diseases, metabolic diseases, immune-mediated disorders, allergic diseases, dermatological diseases, malignant diseases, gastrointestinal disorders, respiratory disorders, cardiovascular disorders, renal disorders, rheumatoid disorders, neurological disorders, endocrine disorders, and aging-related disorders or diseases.

[0033] In one embodiment, the invention provides a short double-stranded DNA (sdDNA) molecule comprising a first strand and a second strand, each strand comprising linked nucleotide monomers selected from the group of nucleotides, their analogs, and modified nucleotides, wherein (a) the first strand is equal in length to the second strand or shorter than the second strand by a number of monomers selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 monomers; (b) the first strand is substantially complementary to a segment of a targeted RNA by at least one targeting region, the first strand being comprised of 8 to 36 (inclusive of both end points of the range) nucleotide monomers linked between adjacent monomers by linkages selected from the group consisting of phosphorothioate linkages, phosphodiester linkages, and mixtures of phosphorothioate and phosphodiester linkages; (c) the second strand is comprised of a first strand having a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 monomers; The present invention provides an sdDNA molecule, wherein the sdDNA molecule is substantially complementary to one strand and forms at least one double-stranded region with the first strand, and the second strand is comprised of 10-36 (both end points of the range included) nucleotide monomers linked between adjacent monomers by linkages selected from the group consisting of phosphorothioate linkages, phosphodiester linkages, and mixtures of phosphorothioate and phosphodiester linkages; (d) the sdDNA molecule comprises at least one ribonucleotide monomer interspersed segment (ISR) linked to at least one deoxyribonucleotide monomer selected from the group consisting of deoxyribonucleotides, analogs thereof, and modified deoxyribonucleotides; and (e) the ISR in the sdDNA molecule comprises at least one ribonucleotide monomer selected from the group consisting of ribonucleotides, analogs thereof, and modified ribonucleotides. In one aspect, the sdDNA molecule is used to modulate target gene expression or function in a cell, e.g., a eukaryotic cell such as a mammalian cell. In a further aspect, the sdDNA molecule is more potent or effective at silencing expression of a target gene in a cell than a corresponding ASO.

[0034] In a second aspect, the present invention provides a pharmaceutical composition, comprising the composition of the first aspect as an active agent and pharma- ceutical acceptable excipient, carrier or diluent.Examples of such carriers 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, polysaccharides, polymers, nanoparticles, nanoemulsions, cholesterol, lipids, lipophilic compounds or moieties, and lipids.

[0035] In a third aspect, the present invention provides a method of using the composition of the first aspect or the pharmaceutical composition of the second aspect to treat or prevent a disease or condition by administering a therapeutically effective amount of an sdDNA molecule of the present invention or a pharmaceutical composition containing such a molecule, the method of administration being a route selected from the group of intravenous injection (iv), subcutaneous injection (sc), orally (po), intramuscular (im) injection, oral administration, inhalation, topical, intrathecal, and other localized administration.

[0036] In one feature, the disease or condition to be prophylactically or therapeutically treated is selected from the group of cancer, autoimmune diseases, inflammatory diseases, degenerative diseases, infectious diseases, proliferative diseases, metabolic diseases, immune-mediated disorders, allergic diseases, dermatological diseases, malignant diseases, gastrointestinal disorders, hepatic disorders, respiratory disorders, cardiovascular disorders, dermatological disorders, renal disorders, rheumatic disorders, neurological disorders, psychiatric disorders, endocrine disorders, and aging-related disorders or diseases.

[0037] In a fourth aspect, the present invention provides a method of using the composition of the first aspect or the pharmaceutical composition of the second aspect to control or modulate gene expression or function in a eukaryotic cell, the method comprising the step of contacting the cell with an effective amount of any of the sdDNA molecules of the present invention or of a pharmaceutical composition containing such a molecule.

[0038] In one embodiment, the contacting step comprises introducing a composition comprising the sdDNA molecule into a target cell in culture or into a target cell in an organism capable of selective gene silencing. In a further embodiment, the introducing step is selected from the group consisting of simple mixing, transfection, lipofection, electroporation, infection, injection, and oral administration, intravenous injection (iv), subcutaneous injection (sc), orally (po), intramuscular (im) injection, inhalation, topical, intrathecal, and other localized administration. In another embodiment, the introducing step comprises using a pharma- ceutically acceptable excipient, carrier, or diluent selected from the group comprising pharmaceutical carriers, lipid nanoparticles, positive charge carriers, liposomes, protein carriers, hydrophobic moieties or molecules, cationic moieties or molecules, GalNAc, polysaccharides, polymers, nanoparticles, nanoemulsions, cholesterol, lipids, lipophilic compounds or moieties, and lipids.

[0039] In certain embodiments, the target gene is an mRNA. In certain embodiments, the target gene is a non-coding RNA, such as a microRNA or lncRNA.

[0040] In some embodiments, the target gene is associated with a disease, a pathological condition or an undesirable condition in a mammal. In a further embodiment, the target gene is a gene of a pathogenic microorganism. In yet a further embodiment, the target gene is a viral gene. In another embodiment, the target gene is a tumor-associated gene. In yet another embodiment, the target gene is a gene associated with a disease selected from the group listed for the third aspect.

[0041] In another aspect, the invention provides an oligomeric duplex comprising (a) one or more deoxyribonucleosides, analogs thereof, or modified deoxyribonucleosides, and (b) one or more ISRs comprising ribonucleosides, analogs thereof, or modified ribonucleosides linked in an antisense sequence of at least 8 nucleobases in length, wherein the antisense sequence is at least 70% complementary to a target sequence.

[0042] Other features and advantages of the present invention are apparent from the additional description provided herein, including different examples. The examples provided illustrate different components and methodologies useful in implementing the present invention. The examples do not limit the claimed invention. Based on this disclosure, one skilled in the art can identify and utilize other components and methodologies useful in implementing the present invention. Although several embodiments have been shown and described, any modifications can be made without departing from the spirit and scope of the present invention. [Brief description of the drawings]

[0043] [Figure 1] FIG. 1A shows an exemplary structure of some embodiments of short double-stranded DNA (sdDNA) according to the principles of the present invention with at least one ribonucleotide interspersed segment (ISR) in the antisense strand (first strand, AS) (and sense strand (second strand, SS)), and an exemplary sequence of sdDNA with a structure designed to target the APOCIII gene. For each duplex depicted here, the sense strand is listed above the antisense strand. FIG. 1B shows the gene silencing efficacy of the sdDNAs shown in FIG. 1A and their corresponding ASOs (having the same structure and sequence as the single-stranded AS of the sdDNAs in FIG. 1A) as a comparison. After transfecting these sdDNAs and the corresponding ASOs at 100 pM into HepaRG cells, the relative mRNA levels of the APOCIII gene were analyzed.

[0044] [Diagram 2]Figure 2A shows the exemplary structure and sequence of some embodiments of symmetric sdDNA of various lengths. Figure 2B shows the gene silencing efficacy of the sdDNA shown in Figure 2A targeting the APOCIII gene. Figure 2C shows the gene silencing efficacy of the corresponding single-stranded AS oligonucleotides having the same sequence as the antisense strand of each sdDNA shown in Figure 2A targeting the APOCIII gene. After transfecting sdDNA and the corresponding single-stranded AS oligonucleotides at 100 pM into HepaRG cells, the relative mRNA level of the APOCIII gene was detected.

[0045] [Diagram 3] Figure 3A shows the exemplary structure and sequence of some embodiments of symmetric sdDNA of various lengths. Figure 3B shows the gene silencing efficacy of targeting APOCIII gene by sdDNA shown in Figure 3A. After transfecting sdDNA into HepaRG cells at 100 pM, detect the relative mRNA level of APOCIII gene.

[0046] [Figure 4] Figure 4A shows the exemplary structure and sequence of some embodiments of sdDNA with different PS modification motifs in SS. Figure 4B shows the gene silencing efficacy of the sdDNA shown in Figure 4A and their corresponding ASO (having the same structure and sequence as the single-stranded AS of sdDNA in Figure 4A) targeting APOCIII gene. After transfecting sdDNA and corresponding ASO into HepaRG cells at 100pM, detect the relative mRNA level of APOCIII gene.

[0047] [Diagram 5]Figure 5A shows the exemplary structure and sequence of some embodiments of sdDNA with various ISR motifs in antisense strand. Figure 5B shows the gene silencing efficacy of APOCIII gene targeted by sdDNA shown in Figure 5A. After transfecting sdDNA into HepaRG cells at 100pM, detect the relative mRNA level of APOCIII gene.

[0048] [Figure 6] Figure 6A shows the exemplary structure and sequence of some embodiments of sdDNA that has at least one mismatch in the antisense strand when binding to target RNA. Figure 6B shows the gene silencing efficacy of targeting APOCIII gene by sdDNA shown in Figure 6A. After transfecting sdDNA into HepaRG cells at 100 pM, detect the relative mRNA level of APOCIII gene.

[0049] [Figure 7] Figure 7A shows the exemplary structure and sequence of some embodiments of sdDNA with SS longer than AS. Figure 7B shows the gene silencing efficacy of the sdDNA shown in Figure 7A and their corresponding ASO (having the same structure and sequence as the single-stranded AS of sdDNA in Figure 7A) targeting APOCIII gene. After transfecting sdDNA and corresponding ASO into HepaRG cells at 100pM, detect the relative mRNA level of APOCIII gene. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] Detailed Description of the Invention The present invention refers to gene or RNA modulation / silencing technology using short double-stranded DNA. This new technology is used to modulate gene expression or function in vitro and in vivo by using short double-stranded DNA (sdDNA) composition. The present invention also provides the method of using the composition to modulate target gene expression or function, or for disease treatment or prevention, and other medical and biological applications. These compositions and methods provide high efficacy in controlling gene expression or gene function, but also reduce dose-dependent toxicity.

[0051] 1.Definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0052] When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below those numerical values. In general, the term "about" is used herein to modify a numerical value by a variance of 20%, 10%, 5% or 1% above and below the stated value. In some embodiments, the term "about" is used to modify a numerical value by a variance of 10% above and below the stated value. In some embodiments, the term "about" is used to modify a numerical value by a variance of 5% above and below the stated value. In some embodiments, the term "about" is used to modify a numerical value by a variance of 1% above and below the stated value.

[0053] As used herein, the term "analog" or "analogous" refers synonymously to functional or structural equivalents.For example, nucleoside and nucleotide analogs have been used in the clinical treatment of cancer and viral infections for decades, and new compounds are continually synthesized and evaluated by researchers and the pharmaceutical industry.See, for example, Jordheim LP et al., Nat Rev Drug Discov 12, 447-464 (2013).

[0054] As used herein, the term "deoxyribonucleoside monomer" refers to a nucleoside monomer, including naturally occurring deoxyribonucleosides, their analogs, and modified deoxyribonucleosides. The term "deoxyribonucleotide monomer" refers to a nucleotide monomer, including naturally occurring deoxyribonucleotides, their analogs, and modified deoxyribonucleotides.

[0055] As used herein, the term "ribonucleoside monomer" refers to a nucleoside monomer, including naturally occurring ribonucleosides, their analogs, and modified ribonucleosides. The term "ribonucleotide monomer" refers to a nucleotide monomer, including naturally occurring ribonucleotides, their analogs, and modified ribonucleotides.

[0056] As used herein, the term "nucleoside" refers to a compound that comprises a nucleic acid base portion and a sugar portion. Nucleoside monomers include, but are not limited to, naturally occurring nucleosides (e.g., deoxyribonucleosides and ribonucleosides as found in DNA and RNA, respectively), their analogs, and modified nucleosides. Nucleoside monomers can be deoxyribonucleoside monomers or ribonucleoside monomers. Nucleoside monomers can be linked to a phosphate portion, for example, to become nucleotide monomers.

[0057] As used herein, the term "nucleotide" refers to a nucleoside that further comprises a phosphate linking group. Nucleotide monomers include, but are not limited to, naturally occurring nucleotides (e.g., deoxyribonucleotides and ribonucleotides as found in DNA and RNA, respectively), their analogs, and modified nucleotides. Nucleotide monomers can be deoxyribonucleotide monomers or ribonucleotide monomers. Modified nucleotides can be modified in one or more of the following: its nitrogenous nucleobase moiety, its 5-carbon sugar moiety, and its phosphate linking group, resulting in a change in internucleoside linkage.

[0058] As used herein, the term "oligo" or "oligonucleotide" refers to a compound that comprises multiple linked nucleoside monomers. In certain embodiments, one or more of the nucleoside monomers or one or more of the internucleoside linkages are modified.

[0059] 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.

[0060] As used herein, the term "double-stranded DNA," as in "short double-stranded DNA (sdDNA)," refers to a molecule composed of two strands or chains of nucleotide monomers that hybridize to each other in the form of a double-stranded oligonucleotide and that is contacted with a cell or administered to a subject, in which the majority of the linked nucleotide monomers of the critical RNA targeting motif, i.e., 50% or more, are deoxyribonucleotide monomers that contain modified deoxyribonucleotides.

[0061] As used herein, the term "motif" refers to a pattern of chemically distinct regions, for example within an antisense or sense strand.

[0062] As used herein, the term "immediately adjacent" means that there are no intervening elements between the two elements, eg, regions, segments, nucleotides and / or nucleosides.

[0063] As used herein, the term "modified nucleotide" means a nucleotide having at least one modified sugar moiety, modified internucleoside linkage, and / or modified nucleobase.

[0064] As used herein, the term "modified nucleoside" means a nucleoside having at least one modified sugar moiety and / or modified nucleobase.

[0065] As used herein, the term "modified oligonucleotide" means an oligonucleotide that contains at least one modified nucleotide.

[0066] As used herein, the term "naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage.

[0067] As used herein, the term "modified internucleoside linkage" refers to a substitution or any change from a naturally occurring internucleoside bond. For example, a phosphorothioate linkage is a modified internucleoside linkage.

[0068] As used herein, the term "natural sugar moiety" means a sugar that is naturally found in DNA (2-H) or RNA (2-OH).

[0069] As used herein, the term "modified sugar" refers to a substitution or change from a natural sugar. For example, a 2'-O-methoxyethyl modified sugar is a modified sugar.

[0070] As used herein, the term "bicyclic sugar" means a furosyl ring modified by bridging two non-geminal ring atoms. A bicyclic sugar is a modified sugar.

[0071] As used herein, the term "bicyclic nucleic acid," "BNA," "bicyclic nucleoside," or "bicyclic nucleotide" refers to a nucleoside or nucleotide in which the furanose portion of the nucleoside or nucleotide contains a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system.

[0072] As used herein, the term "2'-O-methoxyethyl" (also 2'-MOE, 2'-O(CH2)2-OCH3 and 2'-O-(2-methoxyethyl)) refers to an O-methoxy-ethyl modification at the 2' position of the furosyl ring. A 2'-O-methoxyethyl modified sugar is a modified sugar. As used herein, the term "2'-O-methoxyethyl nucleotide" (also 2'-MOE RNA) refers to a modified nucleotide that contains a 2'-O-methoxyethyl modified sugar moiety.

[0073] As used herein, the term "modified nucleobase" refers to any nucleobase other than adenine, cytosine, guanine, thymidine or uracil.For example, 5-methylcytosine is a modified nucleobase.On the other hand, as used herein, "unmodified nucleobase" refers to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

[0074] As used herein, the term "5-methylcytosine" means a cytosine modified with a methyl group attached to position 5. For example, 5-methylcytosine is a modified nucleobase.

[0075] As used herein, "RNA-like nucleotides" refers to modified nucleotides that, when incorporated into an oligonucleotide, adopt a Northern configuration and function like RNA. RNA-like nucleotides include, but are not limited to, 2'-endofuranosyl nucleotides, bridged nucleic acids (BNA), LNA, cEt, 2'-O-methylated nucleic acids, 2'-O-methoxyethylated (2'-MOE) nucleic acids, 2'-fluorinated nucleic acids, 2'-O-aminopropylated (2'-AP) nucleic acids, hexitol nucleic acids (HNA), cyclohexane nucleic acids (CeNA), peptide nucleic acids (PNA), glycol nucleic acids (GNA), threose nucleic acids (TNA), morpholino nucleic acids, tricyclo-DNA (tcDNA) and RNA alternatives.

[0076] As used herein, "DNA-like nucleotides" refers to modified nucleotides that function like DNA when incorporated into an oligonucleotide. DNA-like nucleotides include, but are not limited to, 2'-deoxy-2'-fluoroarabinose (FANA) nucleotides and DNA surrogates.

[0077] As used herein, "non-coding RNA" refers to an RNA molecule that is not translated into a protein. Examples of non-coding RNA include transfer RNA (tRNA) and ribosomal RNA (rRNA), as well as small non-coding RNA and long ncRNA (lncRNA). As used herein, examples of "small non-coding RNA" include, but are not limited to, microRNA (miRNA), asRNA, pre-miRNA, pri-miRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, and mimics of any of the foregoing. As used herein, "lncRNA", "long non-coding RNA" is a transcribed RNA molecule that does not code for a protein and contains more than 200 nucleotides. lncRNA can also be subject to common post-transcriptional modifications, including 5'-capping, 3'-polyadenylation, and splicing. In general, IncRNAs are a diverse class of molecules that play various roles in modifying genes and genome functions. For example, it is known that lncRNAs control gene transcription, translation, and epigenetic regulation. Examples of IncRNA include, but are not limited to, Kcnqlotl, Xlsirt, Xist, ANRIL, NEAT1, NRON, DANCR, OIP5-AS1, TUG1, CasC7, HOTAIR and MALAT1. As used herein, "splice" or "splicing" refers to the natural process of removing unnecessary regions of RNA and reforming the RNA. An example of the modulation of RNA target function by the oligonucleotide comprising the duplex is the modulation of non-coding RNA function. In some embodiments, the oligonucleotide or oligonucleotide duplex is designed to target one of the small non-coding RNAs described above. In some embodiments, the oligonucleotide or oligonucleotide duplex is designed to target miRNA. In some embodiments, the oligonucleotide or oligonucleotide duplex is designed to target pre-miRNA.In some embodiments, the oligonucleotide or oligonucleotide duplex is designed to target pri-miRNA.In some embodiments, the oligonucleotide or oligonucleotide duplex is designed to target lncRNA.In some embodiments, the oligonucleotide or oligonucleotide duplex is designed to target splice.

[0078] The terms "isolated" or "purified" as used herein refer to a material that is substantially or essentially free from components with which it is normally associated in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

[0079] The term "interspersed" as used herein refers to having different types of moieties in adjacent spaces, e.g., having different modifications to the same type of nucleotide or nucleotide analog beside different types of nucleotides or nucleotide analogs. In various embodiments of the present invention, an "interspersed segment of ribonucleotide monomer" refers to a segment of an oligonucleotide strand in which one or more ribonucleotides are connected to at least one moiety that is a different type from the ribonucleotide. For example, when the ribonucleotide is unmodified, the different type of moiety can be a deoxynucleotide or analog thereof, a modified deoxynucleotide, a modified ribonucleotide, or a ribonucleotide analog. When the ribonucleotide is modified, the different type of moiety is a deoxynucleotide or analog thereof, a modified deoxynucleotide, an unmodified ribonucleotide, a differently modified ribonucleotide, or a different type of ribonucleotide analog.

[0080] As used herein, "modulating", "regulating" and its grammatical equivalents refer to either increasing or decreasing (e.g., silencing), in other words, either up-regulating or down-regulating.As used herein, "gene silencing" refers to the reduction of gene expression, and can refer to the reduction of gene expression of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the targeted gene.

[0081] As used herein, the terms "inhibiting", "to inhibit" and their grammatical equivalents, when used in the context of a biological activity, refer to the downregulation of the biological activity, which may reduce or eliminate the targeted function, such as the production of a protein or the phosphorylation of a molecule. In certain embodiments, inhibition may refer to a reduction of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the targeted activity. When used in the context of a disorder or disease, these terms refer to the success of preventing the onset of symptoms, alleviating symptoms, or eliminating a disease, condition or disorder.

[0082] As used herein, the term "substantially complementary" or "complementary" refers to complementarity in the base-paired double-stranded region between the two strands of linked nucleosides, without any single-stranded regions, such as terminal overhangs, or gap regions between the two double-stranded regions. Complementarity does not need to be perfect, for example, there may be any number of base pair mismatches between the two strands of linked nucleosides. However, if the number of mismatches is so large that no hybridization occurs even under minimally stringent hybridization conditions, the sequence is not a substantially complementary sequence. Specifically, when two sequences are referred to herein as "substantially complementary," it means that the sequences are sufficiently complementary to each other to hybridize under the selected reaction conditions. The relationship between nucleic acid complementarity and the stringency of hybridization sufficient to achieve specificity is well known in the art. Two substantially complementary strands can be, for example, completely complementary, or can contain one to many mismatches, provided that the hybridization conditions are sufficient to allow, for example, discrimination between matched and unmatched sequences. Thus, substantially complementary sequences can refer to sequences that have at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% base pair complementarity in the double-stranded region, or any number in between.

[0083] As used herein, "fully complementary" or "100% complementary" means that each nucleobase of the nucleobase sequence of the first strand of linked nucleosides has a complementary nucleobase in the second nucleobase sequence of the second strand of linked nucleosides.In certain embodiments, the first strand of linked nucleosides is an antisense compound and the second strand of linked nucleosides is a target nucleic acid.In certain embodiments, the first strand of linked nucleosides is a sense compound and the second strand of linked nucleosides is an antisense compound, or vice versa.

[0084] As used herein, the term "targeting region" refers to the region in an oligonucleotide strand, which is substantially or completely complementary to another oligonucleotide strand, and therefore these two strands hybridize or anneal with each other at such targeting region under suitable conditions.For example, antisense strand can contain targeting region, and can hybridize with target mRNA.

[0085] The terms "administer", "administering" or "administration" are used herein in their broadest sense. These terms refer to any method of introducing a compound or pharmaceutical composition described herein into a subject, and may include, for example, introducing a compound into a subject systemically, locally, or in situ. Thus, compounds of the present disclosure produced in a subject from a composition (whether or not the composition contains the compound) are encompassed by these terms. When these terms are used in conjunction with the terms "systemic" or "systemically", they generally refer to the in vivo systemic absorption or accumulation of a compound or composition in the bloodstream, followed by distribution throughout the entire body.

[0086] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or pharmaceutical compound described herein that is sufficient to affect an intended outcome, including but not limited to disease treatment, as described below. In some embodiments, a "therapeutically effective amount" is an amount that is effective 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 administered systemically, locally, or in situ (e.g., the amount of 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 condition to be treated, such as the subject's weight and age, the severity of the condition, or the mode of administration, and can be easily determined by one of skill in the art. The term also applies to a dose that will induce a specific response in a target cell, such as reduced cell migration. The specific dose may vary depending, for example, on the particular pharmaceutical composition, the subject and their age and pre-existing health conditions or risk of health conditions, the dosing regimen to be followed, the severity of the disease, whether it is administered in combination with other agents, the timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

[0087] The term "cancer" in a subject refers to the presence of cells that have characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain morphological features. Often, cancer cells will be present in the form of a tumor or mass, but such cells may be present alone in a subject or circulate in the bloodstream as independent cells, such as leukemia or lymphoma cells. Examples of cancer as used herein include, but are not limited to, lung cancer, pancreatic cancer, bone cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, breast cancer, uterine cancer, ovarian cancer, peritoneal cancer, colon cancer, rectal cancer, colorectal adenocarcinoma, anal cancer, stomach cancer, gastric cancer, gastrointestinal cancer, gastric adenocarcinoma, adrenocorticoid cancer, and the like. carcinoma, uterine cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, gastroesophageal junction cancer, gastroesophageal adenocarcinoma, chondrosarcoma, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, Ewing's sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, testicular cancer, ureteral cancer, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, biliary tract cancer, kidney cancer, renal cell carcinoma, chronic or acute leukemia, lymphocytic lymphoma, central nervous system (CNS) neoplasms, spinal axis tumor, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, including refractory versions of any of the foregoing cancers, or combinations of one or more of the foregoing cancers. Some of the exemplified cancers are included in the general term and are included in this term. For example, the general term urological cancer includes bladder cancer, prostate cancer, kidney cancer, testicular cancer, etc., and another general term hepatobiliary cancer includes liver cancer (which is itself a general term that includes hepatocellular carcinoma or biliary tract cancer), gallbladder cancer, biliary tract cancer, or pancreatic cancer. Both urological cancer and hepatobiliary cancer are contemplated by the present disclosure and are included in the term "cancer".

[0088] The term "pharmaceutical composition" is a formulation containing an active ingredient, such as a molecule or composition disclosed herein, in a form suitable for administration to a subject, often in admixture with other substances, such as a pharmaceutical carrier, such as a sterile aqueous solution. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial. The amount of active ingredient in a unit dose of the composition is an effective amount and varies according to the particular treatment involved. Those skilled in the art will understand that routine variations in dosage are sometimes necessary depending on the age and condition of the patient. The dosage will also depend on the route of administration. Various routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, and the like. Dosage forms for topical or transdermal administration of the sdDNA of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants.

[0089] The term "pharmaceutical agent" means a substance that provides a therapeutic benefit when administered to an individual.

[0090] The term "pharmaceutical acceptable carrier" refers to a medium or diluent that does not interfere with the structure of a compound.Some of such carriers allow pharmaceutical compositions to be formulated, for example, as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and lozenges for oral ingestion by subjects.Some of such carriers allow pharmaceutical compositions to be formulated for injection, infusion or local administration.For example, a pharmaceutical acceptable carrier is a sterile aqueous solution.

[0091] The term "pharmaceutically acceptable derivatives" includes derivatives of the compounds described herein, such as solvates, hydrates, esters, prodrugs, polymorphs, isomers, isotopically labeled variants, pharmaceutically acceptable salts, and other derivatives known in the art.

[0092] The term "pharmaceutically acceptable salt" refers to a physiologically and pharma- ceutically acceptable salt of a compound, i.e., a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects thereto. The term "pharmaceutically acceptable salt" or "salt" includes salts prepared by reaction of the parent compound with pharma- ceutically acceptable non-toxic acids or bases, including inorganic or organic acids and bases. The pharma- ceutically acceptable salts of the compounds described herein can be prepared by methods well known in the art. For a review of pharma-ceutically acceptable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use (Wiley-VCH, Weinheim, Germany, 2002). Pharmaceutically acceptable salts can include, but are not limited to, acid addition salts, including hydrochloride, hydrobromide, phosphate, sulfate, hydrogen sulfate, alkylsulfonate, arylsulfonate, acetate, benzoate, citrate, maleate, fumarate, succinate, lactate and tartrate; alkali metal cations, such as Na, K, Li, alkaline earth metal salts, such as Mg or Ca, or organic amine salts.In particular, the sodium salt of oligonucleotides has been proven to be useful and is well tolerated for therapeutic administration to humans.Therefore, in one embodiment, the compounds described herein are in the form of sodium salt.

[0093] As used herein, the term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., that will be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to human subjects.

[0094] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" as used herein refer to both (1) therapeutic measures that cure, slow, reduce symptoms, and / or halt the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent or slow the onset of the targeted pathological condition or disorder. Thus, those in need of treatment include those who already have the disorder; those who are susceptible to the disorder; and those in whom disease is to be prevented. A subject is successfully "treated" according to the methods of the present invention if the patient exhibits one or more of the following: a reduction in the number of cancer cells or a complete absence of cancer cells; a reduction in tumor size; inhibition or absence of cancer cell invasion into peripheral organs, including the spread of cancer to soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; relief of one or more symptoms associated with a particular cancer; reduced morbidity and mortality; and improved quality of life.

[0095] The term "carrier" as used herein means a pharma- ceutically acceptable material, composition or vehicle, such as, for example, a liquid or solid filler, diluent, excipient, solvent or encapsulating material, that is involved in or capable of carrying or transporting a subject pharmaceutical compound from one organ or part of the body to another organ or part 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 pharma- ceutically acceptable carriers, carriers, and / or diluents include sugars such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances utilized in pharmaceutical formulations. Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate, magnesium stearate, and polyethylene oxide-polypropylene oxide copolymers, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives, and antioxidants can also be present in the composition. 2. Certain embodiments—sdDNA structural scaffolds

[0096] Certain embodiments of the present invention provide a duplex composition in which both the antisense strand and the sense strand are composed of linked nucleoside monomers. Fifty percent or more of the nucleoside monomers in the essential RNA targeting motif are deoxyribonucleoside monomers, or fifty percent or more of the nucleobases in one strand of the double-stranded region of the sdDNA molecule comprise deoxyribonucleoside monomers, and some of the deoxyribonucleoside monomers and / or internucleoside linkages contained therein may be modified from those found in natural DNA. The duplex DNA molecule of the present invention further comprises ribonucleoside monomers in one or more ribonucleotide monomer interspersed segments ("ISRs"). One or more ISRs may be found in either the antisense strand or the sense strand, or in both. In some embodiments, each ISR is composed of one ribonucleotide monomer, or two, three, four, five, or six consecutive ribonucleotide monomers, independent of each other. In some embodiments, the ISR has at least two consecutive linked ribonucleotide monomers.

[0097] Both the antisense and sense strands of the short double-stranded DNA (sdDNA) of the present invention are relatively short, and the sense strand is at least equal in length to the antisense strand, and therefore is a "short double-stranded DNA" (sdDNA). Moreover, in certain embodiments, the length is the same between the two strands, and thus the double-stranded molecule of the present invention may be more specifically referred to as a "symmetric short double-stranded DNA."

[0098] Exemplary structures and sequences of duplex molecules of the invention are shown in Figures 1A, 2A, 4A, 5A, 6A and 7A, where the ISR is found in both strands or only in the antisense strand.

[0099] In some embodiments, the antisense and sense strands of the same length form a symmetric structure without any overhangs.

[0100] The compositions of the present invention are used to modulate gene expression or function in eukaryotic cells in at least three ways: (i) contacting a cell with one type of sdDNA molecule or administering it to a subject; (ii) contacting a cell with different types of sdDNA molecules with a cell or administering it to a subject separately at different times; and (ii) contacting a cell with different types of sdDNA molecules with a cell or administering it to a subject simultaneously.

[0101] In certain embodiments, the antisense oligonucleotide strand comprises a nucleobase sequence region, 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 the target segment of the target gene to which it is targeted, including mRNA and non-coding RNA. In certain embodiments, the antisense oligonucleotide has a nucleobase sequence that comprises a completely complementary sequence of the target segment of the target gene to which it is targeted. In certain embodiments, the antisense oligonucleotide has a nucleobase sequence that contains at most one, two, or three mismatches when hybridized to the target segment of the target RNA to which it is targeted. In certain embodiments, the target gene is selected from mRNA or non-coding RNA involved in mammalian disease. In some embodiments, at least one ISR is located within the targeting region of the antisense strand. In certain embodiments, the ISR is located at or near the 5' end of the antisense strand (i.e., within one third of the length of the strand, which means, for example, within 7 nucleobases for a strand that is about 21 nucleobases long, counting from its ends). Alternatively, the ISR is located at or near the 3' end of the antisense strand (i.e., within one third of the length of the strand, which means, for example, within 7 nucleobases for a strand that is about 21 nucleobases long, counting from its ends). In some embodiments, the ISR, or at least a portion of the ISR, is also located in the more central part of the antisense strand, i.e., in the middle third of the length, which means, for example, more than 7 nucleobases away from both ends of the antisense strand for a strand that is about 21 nucleobases long.

[0102] In various embodiments, the first or 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 thereof, or a range bounded by any two of the foregoing values ​​(including both endpoints of the range). For example, some length ranges of the first antisense strand include: 8 to 50 nucleotide monomers; 8 to 36 nucleotide monomers; 8 to 33 nucleotide monomers; 10 to 36 nucleotide monomers; 10 to 30 nucleotide monomers; 10 to 29 nucleotide monomers; 12 to 36 nucleotide monomers; 12 to 29 nucleotide monomers; 12 to 28 nucleotide monomers; 12 to 26 nucleotide monomers; 12 to 25 nucleotide monomers; 13 to 25 nucleotide monomers; 13 to 24 nucleotide monomers; 13 to 23 nucleotide monomers; 14 to 24 nucleotide monomers; 15 to 23 nucleotide monomers; and 16 to 23 nucleotide monomers; and at least 8 nucleotide monomers.

[0103] In certain embodiments, the antisense oligonucleotide strand is 10-36 (inclusive) nucleotide monomers in length. In other words, the antisense strand is 10-36 (inclusive) linked nucleobase monomers. In other embodiments, the antisense strand comprises a modified oligonucleotide consisting of 8-100, 10-80, 12-50, 14-30, 15-23, 16-22, 16-21, or 20 (inclusive) linked nucleobases.

[0104] In certain embodiments, the antisense oligonucleotide consists of 14 to 23 linked nucleoside monomers (inclusive of both endpoints of the range). In certain embodiments, the antisense oligonucleotide consists of 20 linked nucleoside monomers. In certain embodiments, the antisense oligonucleotide consists of 16 linked nucleoside monomers.

[0105] In certain embodiments, the sense strand is substantially complementary to the antisense strand and comprises a nucleobase sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to the sequence of the linked region of the antisense oligonucleotide, measured over the entire nucleobase sequence of the antisense strand. These substantially complementary sequences from both strands form one or more double-stranded regions. In certain embodiments, sense strand has a nucleic acid base sequence that comprises the complete complement of the linked region of antisense strand.As a result, the two strands form a double-stranded region that comprises 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, at least one ISR is located in the double-stranded region of sense strand.

[0106] In certain embodiments, ISR is located at or near the 5' end of sense strand (within 33% of the total number of nucleobases counting from end to end) or at or near the 3' end of sense strand (within 33% of the total number of nucleobases counting from end to end).In some embodiments, ISR, or at least a part of ISR, is also located in the more central part of sense strand, i.e., located more than 33% of the total number of nucleobases away from both ends of sense strand.In some embodiments, ISR does not need to be located in sense strand.

[0107] In certain features, the sense oligonucleotide strand has a length equal to or greater than the antisense oligonucleotide strand. In certain embodiments, the sense strand has a length from full length to 16 linked nucleobases long, longer than the antisense strand. In certain embodiments, the sense strand is 6-66 (inclusive) nucleotide monomers in length. In other words, these sense strands are 6-66 (inclusive) linked nucleobases. In other embodiments, the sense strand comprises an oligonucleotide consisting of 20-21, 8-36, 10-30, 12-25, 14-24, or 16-23 (inclusive) linked nucleobases. In certain such embodiments, the sense strand comprises an oligonucleotide 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, or 66 linked nucleobases in length, or a range defined by any two of the foregoing values ​​(including both endpoints of the range). In some embodiments, the sense strand is a sense oligonucleotide.

[0108] In one feature, the length of the second sense strand is equal to that of the first antisense strand.In certain embodiments, both ends of the duplex are blunt-ended.In other features, the second strand or sense strand has a backbone length longer than the first strand or antisense strand by at least the following number of nucleotide monomers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19 and 20. In various embodiments, the second or 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 thereof, or a range bounded by any two of the foregoing values ​​(including both endpoints of the range). In certain embodiments, for example, a portion of the range of the second, sense strand includes a tide of 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; a tide of 16-23 nucleotide monomers; and at least 8 nucleotide monomers.

[0109] 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 is comprised of 8-36 linked nucleoside monomers (inclusive of both end points of the range). In certain embodiments, the sense strand is comprised of 13 linked nucleoside monomers. In certain embodiments, the sense strand is comprised of 14 linked nucleoside monomers.

[0110] In some embodiments of the present invention, the two strands of the sdDNA molecule form a symmetric duplex without any overhang.In some other various embodiments, the two ends of the first strand (antisense strand) are in one of the following configurations: 3'-overhang and 5' blunt end; 5'-overhang and 3' blunt end; 3' overhang and 5' recessed end; 3' recessed end and 5' overhang; or 3' recessed end and 5' recessed end.

[0111] 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 1-8, 1-5, 1-3, or 1-2 nucleotide monomers (including both endpoints of the ranges).

[0112] 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 3'-overhang of the sense strand has a length of 1 to 8, 1 to 5, 1 to 3, or 1 to 2 nucleotide monomers (including both endpoints of the ranges).

[0113] In the sdDNA molecule of the present invention, at least one nucleotide monomer in the first strand and / or the second strand can be a modified nucleotide or nucleotide analog, such as a sugar-modified nucleotide, a backbone-modified nucleotide, and / or a base-modified nucleotide. In some embodiments, such a backbone-modified nucleotide has at least a modification of the internucleoside linkage, for example, to include at least one of a nitrogen or sulfur heteroatom. In some embodiments, the modified internucleoside linkage is or includes a phosphorothioate (P=S) group, a phosphotriester, a methylphosphonate, or a phosphoramidate.

[0114] In certain embodiments, the antisense strand and / or the sense strand comprises at least one modified internucleoside linkage. Such modified internucleoside linkage can be between two deoxyribonucleoside monomers, between two ribonucleoside monomers, or between one deoxyribonucleoside monomer and one ribonucleoside monomer. Alternatively, the phosphate group on at least one of the terminal nucleoside monomers can be modified. In certain embodiments, the internucleoside linkage is a phosphorothioate internucleoside linkage. In certain embodiments, the internucleoside linkage is a thiophosphoramidate internucleoside linkage. In certain embodiments, each internucleoside linkage of the oligonucleotide chain is a phosphorothioate internucleoside linkage. In certain embodiments, all of the internucleoside linkages within a strand (antisense or sense or both) are phosphorothioate internucleoside linkages, or a mixture of phosphorothioate and phosphodiester linkages.

[0115] In certain embodiments, the antisense strand and / or the sense strand comprises at least one nucleoside monomer having a modified sugar moiety. Such a nucleoside monomer may be a deoxyribonucleoside monomer or a ribonucleoside monomer.

[0116] In certain embodiments, the 2' position of the modified sugar moiety is replaced with a group selected from OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, where each R is independently C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I. In some embodiments, the 2' position of the modified sugar moiety is replaced with a group selected from allyl, amino, azido, thio, O-allyl, O-C1-C 10 Alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), O-CH2-C(=O)-N(R m )(R n ), or O-CH2-C(=O)-N(R1)-(CH2)2-N(R m )(R n ), where each R l , R m and R n are independently H or substituted or unsubstituted C1-C 10 In some embodiments, the modified sugar moiety is selected from the group of 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, 2'-OCH2CH3, 2'-OCH2CH2F, and 2'-O(CH2)2OCH3 substituents. In some embodiments, the modified sugar moiety is substituted with a bicyclic sugar selected from the group of 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (cEt), and 4'-CH(CHOCH3)-O-2', 4'-C(CH3)(CH3)-O-2', 4'-CH2-N(OCH3)-2', 4'-CH2-ON(CH3)-2', 4'-CH2-N(R)-O-2' (wherein R is H, C1-C12 alkyl, or a protecting group), 4'-CH2-C(H)(CH3)-2', and 4'-CH2-C-(=CH2)-2'.

[0117] In some embodiments, the modified sugar moiety is selected from the group of 2'-O-methoxyethyl modified sugars (MOE), 4'-(CH2)-O-2' bicyclic sugars (LNA), 2'-deoxy-2'-fluoroarabinose (FANA), and methyl(methyleneoxy)(4'-CH(CH3)-O-2) bicyclic sugars (cEt).

[0118] In some embodiments, the antisense and / or sense strands of the molecules of the invention comprise at least one nucleotide monomer having a modified nucleobase. Such nucleoside monomers may be deoxyribonucleoside monomers or ribonucleoside monomers.

[0119] In some embodiments, modified nucleobases are 5-methylcytosine (5-Me-C), inosine bases, tritylated bases, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine; 5-propynyl (-C≡C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases; 6-azouracil, cytosine, and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, and 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0120] In certain embodiments, the modified nucleobase in the molecule of the present invention is 5-methylcytosine.In some embodiments, each cytosine base in the molecule of the present invention is 5-methylcytosine.In some embodiments, the modified nucleobase is 5-methyluracil.In some embodiments, each uracil is 5-methyluracil.

[0121] In certain embodiments, either or both of the antisense or sense strands of the molecules of the invention comprise linked deoxynucleoside monomers. In certain embodiments, the entire sense strand is exclusively composed of linked deoxynucleoside monomers. In one feature, either the antisense strand or both the antisense and sense strands further comprise an ISR consisting of one or more linked ribonucleoside monomers in addition to linked deoxynucleoside monomers. In addition, there may be even more ISR segments. The ISR may be anywhere in either strand. In some embodiments, one or more ISRs comprise a terminal nucleoside monomer or a penultimate terminal nucleoside monomer. In certain embodiments, each of the ISRs, independently of each other, consists of one ribonucleoside monomer, or two, three, four, or five linked ribonucleoside monomers.

[0122] In certain embodiments, at least half of the nucleobases in at least one strand of the double-stranded region are deoxyribonucleotide monomers.

[0123] In certain embodiments, at least 50% of the nucleotides in the first strand in the RNA-targeting portion of the double-stranded region are deoxyribonucleotide monomers.

[0124] In certain embodiments, the total number of ribonucleotide monomers in an sdDNA molecule is at most the total number of deoxyribonucleotide monomers in the same sdDNA molecule.

[0125] In certain embodiments, at least one or each of the linked ribonucleoside monomers of the ISR is a modified ribonucleotide or ribonucleotide analog. The ribonucleotides may be modified in the same or similar manner as follows: having modified internucleoside linkages, modified sugar moieties, and / or modified nucleobases.

[0126] In some embodiments, the sugar moiety of the deoxyribonucleotide monomer is either a sugar moiety of a naturally occurring deoxyribonucleotide (2-H) or 2'-deoxy-2'-fluoroarabinose (FANA).

[0127] In some embodiments, the sugar moiety of the ribonucleotide monomer is selected from the group consisting of naturally occurring ribonucleotides (2-OH), 2'-F modified sugars, 2'-OMe modified sugars, 2'-O-methoxyethyl modified sugars (MOE), 4'-(CH2)-O-2' bicyclic sugars (LNA), and methyl(methyleneoxy) (4'-CH(CH3)-O-2) bicyclic sugars (cEt).

[0128] In certain embodiments, at least one or each ribonucleoside monomer of each ISR therein in the antisense strand, the sense strand, or both strands has a modified sugar moiety selected from the group of 2'-O-methoxyethyl modified sugar (MOE), 4'-(CH2)-O-2' bicyclic sugar (LNA), and methyl(methyleneoxy)(4'-CH(CH3)-O-2) bicyclic sugar (cEt). In certain embodiments, at least one deoxyribonucleoside monomer in the antisense strand, the sense strand, or both strands has a modified sugar moiety of 2'-deoxy-2'-fluoroarabinose (FANA). In certain embodiments, each ribonucleoside monomer of each ISR has a 2'-O-methoxyethyl modified sugar, a 4'-(CH2)-O-2' bicyclic sugar, or a methyl(methyleneoxy)(4'-CH(CH3)-O-2) bicyclic sugar (cEt), each cytosine is a 5-methylcytosine, each uracil is a 5-methyluracil, or a methyl-pseudouracil, and each internucleoside linkage is a phosphorothioate linkage.

[0129] In certain embodiments, the molecules of the invention have either the antisense or sense strands comprised of deoxynucleoside monomers in which each internucleoside linkage is a phosphorothioate linkage. In certain embodiments, the molecules of the invention have either the antisense or sense strands comprised of deoxynucleoside monomers in which each internucleoside linkage is a natural phosphate linkage without phosphorothioate modifications.

[0130] In certain embodiments, a molecule of the invention comprises a sense strand, in which each nucleotide monomer of the sense strand contains the same modification as the complementary nucleotide monomer of the antisense strand.

[0131] Exemplary structures and exemplary sequences of exemplary molecules of the invention having antisense and sense oligonucleotide strands are shown in Figures 1A, 2A, 4A, 5A, 6A and 7A.

[0132] In certain embodiments, the short DNA duplex and at least one ISR in the antisense strand of the duplex molecule enable strong gene silencing. The data presented in all examples below suggest that the new platform technology based on a duplex with an antisense oligodeoxyribonucleotide, which has at least one ISR in the antisense oligodeoxyribonucleotide, enables extremely strong gene silencing. Further research was conducted on the SAR (structure-activity relationship) characteristics of sdDNA, including various ISR motifs, various lengths, complementarity and mismatches, various modifications, etc. This research helps to define various structural factors and scaffolding features that can affect gene silencing activity. Such SAR factors and design features are important for the design of optimized gene silencers that target various sequences and structures of more than 100,000 different mRNAs in typical mammalian cells, and even more non-coding RNAs. Our data on gene silencing activity and SAR of sdDNA suggest that the gene silencing characteristics of sdDNA are significantly different from siRNA and ASO, indicating a novel and distinct mechanism of gene silencing that has yet to be identified.

[0133] In certain embodiments, the molecules of the present invention can be stabilized against degradation by at least one chemical modification or secondary structure.Sense and antisense oligonucleotide chains can have unmatched or incompletely matched nucleotide monomers.Sense and / or antisense oligonucleotide chains can have one or more nicks (cuts in the nucleic acid backbone), gaps (fragmented chains with one or more missing nucleotides), and modified nucleotides or nucleotide analogs.Not only can any or all of the nucleotide monomers in the sense and antisense oligonucleotide chains be chemically modified, but each chain can be conjugated to one or more moieties or ligands, such as a moiety or ligand selected from peptides, antibodies, antibody fragments, polymers, polysaccharides, lipids, hydrophobic moieties or molecules, cationic moieties or molecules, lipophilic compounds or moieties oligonucleotides, cholesterol, GalNAc, and aptamers, to enhance its functionality.

[0134] In certain embodiments, the double-stranded region of the duplex molecule of the present invention does not contain any mismatches or bulges, and the two strands are completely complementary to each other in the double-stranded region. In another embodiment, the double-stranded region of the duplex contains mismatches and / or bulges.

[0135] In certain embodiments, the target is mRNA or non-coding RNA involved in mammalian disease.In certain embodiments, the target is mRNA.In certain embodiments, the target is non-coding RNA, such as microRNA and lncRNA.Antisense strand can occupy the target by hybridizing to the target sequence if they are substantially complementary to each other, and inactivate the target gene.

[0136] 3. Unmatched or mismatched regions The complementary region between the antisense strand and the sense strand of the present invention may have at least one unmatched region or an incompletely matched region, for example, containing one or more mismatches.In some embodiments, the sense strand of the sdDNA provided by the present invention can contain three or more mismatches (at least 15% of the targeting region) without any effect on the gene silencing activity of the sdDNA.Mismatches in the sense strand may be desirable to reduce off-target effects or allow other features to the sdDNA.

[0137] As known to those skilled in the art, it is possible to introduce mismatched bases without losing activity.Similarly, the strand of the antisense oligonucleotide of the sdDNA of the present invention can contain unmatched or mismatched regions.In some embodiments, the strand of the antisense oligonucleotide of the sdDNA of the present invention can tolerate at least three mismatches (at least 15% of the targeting region) while maintaining gene silencing activity.Mismatches in the antisense strand may be desirable to reduce off-target effects or allow other features to the sdDNA. 4. Modifications

[0138] Nucleoside monomers are base-sugar compositions. The nucleobase (also known as base) portion of a nucleoside monomer is usually a heterocyclic base moiety. Nucleotide monomers are nucleoside monomers that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For nucleoside monomers that include a pentofuranosyl sugar, the phosphate group may be linked to the 2', 3', or 5' hydroxyl portion of the sugar. Oligonucleotides are formed by covalently linking adjacent nucleoside monomers to each other to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.

[0139] Modifications of the sdDNA molecules, antisense strands and / or sense strands of the present invention include substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified sdDNA, antisense strands and / or sense strands are preferred over native forms in some cases due to desirable properties, such as increased inhibitory activity, enhanced cellular uptake, enhanced strand affinity, solubility, reduced non-specific interactions, and resistance to RNase degradation or otherwise enhanced stability. Thus, comparable results can often be obtained with shorter antisense strands having such chemically modified nucleoside monomers. One or more of the natural nucleotides in the antisense and sense strands of the present invention can be replaced with modified nucleotides or nucleotide analogs. Substitutions can be made anywhere in the antisense and sense strands.

[0140] Modifications of oligonucleotide molecules have been investigated to improve the stability of various oligonucleotide molecules, including antisense oligonucleotides, ribozymes, aptamers, and RNAi (Chiu and Rana, 2003; Czauderna et al., 2003; de Fougerolles et al., 2007; Kim and Rossi, 2007; Mack, 2007; Zhang et al., 2006; Schrnidt, 2007; Setten RL et al., 2020; Crooke ST et al., 2018; and Roberts TC et al., 2020).

[0141] Any stabilizing modification known to those skilled in the art can be used to improve the stability of oligonucleotide molecules.In oligonucleotide molecules, chemical modifications can be introduced into phosphate backbone (e.g., phosphorothioate linkage), sugar (e.g., locked nucleic acid, glycerol nucleic acid, cEt, 2'-MOE, 2'-fluorouridine, 2'-O-methyl), and / or base (e.g., 2'-fluoropyrimidine).

[0142] Some examples of such chemical modifications are summarized in the following sections.

[0143] In various embodiments, the modified nucleotides or nucleotide analogs are sugar-, backbone-, and / or base-modified nucleotides.

[0144] 4.1 Modified Internucleoside Linkages or Backbone Modified Nucleotides The naturally occurring internucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester linkage. The sdDNA molecules of the invention having one or more modified, i.e., non-naturally occurring, internucleoside linkages in one or both strands may be chosen over corresponding molecules having only naturally occurring internucleoside linkages because of desirable properties, such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0145] The oligonucleotide chain with modified internucleoside linkage includes not only the internucleoside linkage that holds phosphorus atom, but also the internucleoside linkage that does not have phosphorus atom.In some embodiments, phosphodiester internucleoside linkage is modified to include at least nitrogen and / or sulfur heteroatom.Representative phosphorus-containing internucleoside linkage includes, but is not limited to, phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, thiophosphoramidate and phosphorothioate.The method of preparing phosphorus-containing and non-phosphorus-containing linkage is well known.

[0146] In one embodiment, the modified nucleotide or nucleotide analog is a backbone-modified nucleotide. The backbone-modified nucleotide may have a modification of the phosphodiester internucleoside linkage. In a further embodiment, the backbone-modified nucleotide is a phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage.

[0147] 4.2 Modified sugar moieties The antisense and / or sense strands of the invention may optionally contain one or more nucleoside monomers in which the sugar group has been modified. Such sugar-modified nucleoside monomers may confer enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the strand. In certain embodiments, the nucleoside monomer comprises a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and 2' substituents), bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNAs), modification of the ribosyl ring oxygen atom with S, N(R) or C(R1)(R2) (where R, R1 and R2 are each independently selected from H, C1-C1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H26, H16, H17, H18, H27, H28, H30, H40, H40, H50, H51, H60, H70, H80, H90, H100, H110, H120, H130, H140, H150, H160, H170, H180, H190, H200, H200, H150, H160, H200, H170, H180, H200, H19 ... 12 Examples of chemically modified sugars include 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO2008 / 101157 published August 21, 2008 for other disclosed 5',2'-disubstituted nucleosides), or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2' position (see US Patent Application Publication No. 2005-0130923 published June 16, 2005), or alternatively 5' substitution of BNA (see PCT International Application WO2007 / 134181 published November 22, 2007 where LNA is substituted, for example, with a 5'-methyl or 5'-vinyl group).

[0148] Examples of nucleoside monomers having modified sugar moieties include, but are not limited to, nucleosides containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, 2'-OCH2CH3, 2'-OCH2CH2F and 2'-O(CH2)2OCH3 substituents. The substituent at the 2' position can be any of allyl, amino, azido, thio, O-allyl, O-C1-C2 ... 10and O-CH-C(=O)-N(Rm)(Rn), and O-CH-C(=O)-N(R1)-(CH2)2-N(Rm)(Rn), where each Rl, Rm, and Rn is independently H or a substituted or unsubstituted C1-C 10 It is an alkyl.

[0149] Bicyclic nucleosides are modified nucleosides having a bicyclic sugar moiety. Examples of bicyclic nucleic acids (BNAs) include, but are not limited to, nucleosides that include a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the sdDNA, antisense strand and / or sense strand provided herein include one or more BNA nucleosides in which the bridge includes one of the following formulas: 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' and 4'-CH(CHOCH3)-O-2' (and analogs thereof, see U.S. Patent No. 7,399,845 issued July 15, 2008); 4'-C(CH3)(CH3)-O-2' (and analogs thereof, see U.S. Patent No. 7,399,845 issued July 15, 2008). see PCT / US2008 / 068922, published as WO / 2009 / 006478, published Jan. 8, 2009; 4'-CH2-N(OCH3)-2' (and its analogs, see PCT / US2008 / 064591, published as WO / 2008 / 150729, published Dec. 11, 2008); 4'-CH2-ON(CH3)-2' (see U.S. Patent Application Publication No. 2004-0171570, published Sep. 2, 2004); 4'-CH2-N(R)-O-2' (wherein R is H, C1-C 12alkyl, or a protecting group) (see U.S. Pat. No. 7,427,672, issued Sep. 23, 2008); 4'-CH2-C(H)(CH3)-2' (see Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C-(═CH2)-2' (and its analogs, see PCT / US2008 / 066154, published as WO2008 / 154401, published Dec. 8, 2008).

[0150] In certain embodiments, the bicyclic nucleoside is: (A) α-L-methyleneoxy (4'-CH2-O-2) BNA (B) β-D-methyleneoxy (4'-CH2-O-2) BNA These include, but are not limited to, (C) ethyleneoxy (4'-(CH2)2-O-2') BNAs, (D) aminooxy (4'-CH2-ON(R)-2') BNAs, (E) oxyamino (4'-CH2-N(R)-O-2) BNAs, (F) methyl (methyleneoxy) (4'-CH(CH3)-O-2) BNAs (also called constrained ethyl or cEt), (G) methylene-thio (4'-CH2-S-2') BNAs, (H) methylene-amino (4'-CH2-N(R)-2') BNAs, (I) methyl carbocyclic (4'-CH2-CH(CH3)-2) BNAs, (J) propylene carbocyclic (4'-(CH2)3-2') BNAs, and (K) vinyl BNAs.

[0151] In certain embodiments, the modified nucleotide or nucleotide analog is a sugar-modified ribonucleotide in which the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, and CN, where each R is independently selected from the group consisting of C1-C6 alkyl, alkenyl, and alkynyl, and halo is selected from the group of F, Cl, Br, and I. In certain embodiments, the sugar-modified ribonucleotide is selected from the group of 2'-OMe modified nucleotides, 2'-F modified nucleotides, 2'-O-methoxyethyl (2'MOE) modified nucleotides, LNA (locked nucleic acid) modified nucleotides, GNA (glycerol nucleic acid) modified nucleotides, and cEt (constrained ethyl) modified nucleotides.

[0152] The chemical modification at the 2' position of ribose, such as 2'-O-methyl purine and 2'-fluoro pyrimidine, which increases the resistance to endonuclease activity in serum, can be adopted to stabilize the molecule of the present invention.The position for introducing the modification should be carefully selected to avoid the significant decrease of the silencing / regulation efficacy of the molecule.In certain embodiments, the first nucleotide monomer adjacent to the 5'-terminal nucleotide monomer of antisense strand is 2'-fluoro-ribonucleotide.

[0153] 4.3 Modified nucleobases The antisense and / or sense strands in sdDNA molecules may also have nucleobase (or base) modifications or substitutions. The nucleobase (or base) modifications or substitutions are structurally distinct from naturally occurring or synthetic unmodified nucleobases, but functionally interchangeable. Both natural and modified nucleobases can participate in hydrogen bonds. Such nucleobase modifications may confer nuclease stability, binding affinity, or some other beneficial biological properties to sdDNA molecules. 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 antisense and sense strands. For example, 5-methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

[0154] Additional modified nucleobases include 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 1-methylpseudouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine; 5-propynyl (-C≡C-CH3) uracil and cytosine, and other alkynyl derivatives of pyrimidine bases; 6-azouracil, cytosine and thymine, 5-uracil. (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0155] Heterocyclic base moiety can include purine or pyrimidine base replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.The nucleobase that is particularly useful for increasing the binding affinity of antisense and sense strands includes 5-substituted pyrimidine, 6-azapyrimidine and N-2, N-6 and O-6 substituted purine, said substituted purine includes 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

[0156] In certain embodiments, the modified nucleotide or nucleotide analog is a base modified nucleotide. In certain embodiments, the modified nucleotide or nucleotide analog has an unusual or modified base. In certain embodiments, the modified base is 5-methylcytosine (5'-Me-C). In certain embodiments, each cytosine is a 5-methylcytosine. In certain embodiments, the modified base is 5-methyluracil (5'-Me-U). In certain embodiments, each uracil is a 5-methyluracil.

[0157] Any modified nucleotide or analogue that may aid in stability or affinity can be made without departing from the spirit and scope of the present invention. Some examples of such chemical modifications are the same as those summarized above.

[0158] 5. Pharmaceutical Compositions In some embodiments, the present invention also provides pharmaceutical formulations comprising the sdDNA of the present invention or its pharma- ceutically acceptable derivatives and at least one pharma- ceutically acceptable excipient or carrier. As used herein, "pharma- ceutically acceptable excipient" or "pharma- ceutically 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., which is incorporated herein by reference. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, may also be used. The use of such media and agents for pharma- ceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the sdDNA molecules, its use in the compositions is contemplated.

[0159] Examples of pharma- ceutically acceptable carriers that can be used with the molecules of the invention include, but are not limited to, pharmaceutical carriers, positively charged carriers, liposomes, lipid nanoparticles, protein carriers, hydrophobic moieties or molecules, cationic moieties or molecules, GalNAc, polysaccharides, polymers, nanoparticles, nanoemulsions, cholesterol, lipids, lipophilic compounds or moieties, and lipids.

[0160] In certain embodiments, the present invention provides a method of treatment comprising administering a therapeutically effective amount of a pharmaceutical composition to a subject in need thereof. In certain embodiments, the pharmaceutical composition is administered by a route selected from the group of intravenous injection (iv), subcutaneous injection (sc), orally (po), intramuscular (im) injection, oral administration, inhalation, topical, intrathecal, and other localized administration. In another embodiment, the therapeutically effective amount is 1 ng to 1 g per day, 100 ng to 1 g per day, or 1 μg to 1000 mg per day.

[0161] Methods for formulation are disclosed in PCT International Application No. PCT / US02 / 24262 (WO03 / 01 1224), 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.

[0162] The sdDNA molecules of the present invention are administered in a suitable dosage form prepared by combining a therapeutically effective amount (e.g., an effective level sufficient to achieve a desired therapeutic effect by inhibiting tumor growth, killing tumor cells, treating or preventing a cell proliferative disorder, etc.) of the sdDNA molecule of the present invention (as an active ingredient) with standard pharmaceutical carriers or diluents according to conventional procedures (i.e., by producing a pharmaceutical composition of the present invention).

[0163] These procedures may include mixing, granulating, and compressing or dissolving the ingredients as appropriate to achieve the desired preparation. In another embodiment, a therapeutically effective amount of the sdDNA molecule is administered in a suitable dosage form without standard pharmaceutical carriers or diluents. In some embodiments, a therapeutically effective amount of the duplex molecule of the present invention is administered in a suitable dosage form. Pharmaceutically acceptable carriers include solid carriers, such as lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, and the like. Exemplary liquid carriers include syrup, peanut oil, olive oil, water, and the like. Similarly, carriers or diluents include time-delay materials known in the art, such as glyceryl monostearate or glyceryl distearate, alone or with wax, eihylcellulose, hydroxypropylmethylcellulose, methylmethacrylate, and the like. Other fillers, excipients, flavorants, and other additives, such as those known in the art, can also be included in pharmaceutical compositions according to the invention.

[0164] The pharmaceutical composition of the present invention can be prepared in a generally known manner, for example, by conventional mixing, dissolving, granulating, dragee making, elutriation, emulsifying, encapsulating, entrapping or lyophilization process.The pharmaceutical composition can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and / or auxiliaries that facilitate the processing of sense oligonucleotides and antisense oligonucleotides into medicament-usable preparations.Of course, suitable formulation depends on the route of administration selected.

[0165] The composition, compound, combination or pharmaceutical composition of the present invention can be administered to a subject in many of the well-known methods currently used for chemotherapy treatment.For example, for the treatment of cancer, the sdDNA molecule of the present invention can be directly injected into a tumor, can be injected into the bloodstream or body cavity, or can be taken orally, or can be applied through the skin using a patch.For the treatment of psoriasis conditions, systemic administration (e.g., oral administration) or topical administration to the affected area of ​​the skin is the preferred administration route.The dose selected should be sufficient to constitute an effective treatment, but not so high as to cause unacceptable side effects.The patient's condition (e.g., cancer, psoriasis, etc.) and health status should be closely monitored during and for a reasonable period after treatment.

[0166] 6.Usefulness 6.1 How to use The present invention provides a method for modulating gene expression or function in a cell or organism. The cell can be a eukaryotic cell, for example, a mammalian cell. The method includes contacting the cell or organism with the sdDNA molecule disclosed herein under conditions that allow selective gene silencing, and mediating the selective gene silencing exerted by the sdDNA molecule on a target nucleic acid that has a sequence portion that is substantially complementary to the antisense strand. The target nucleic acid can be an RNA, such as an mRNA or a non-coding RNA, and such an RNA encodes a protein involved in a disease or controls a part of a biological pathway involved in a disease.

[0167] In an embodiment, the contacting step includes introducing the sdDNA molecule into a target cell in culture or into a target cell in an organism capable of selective gene silencing. In a further embodiment, the introducing step includes mixing, transfection, lipofection, infection, electroporation, or other delivery techniques. In another embodiment, the introducing step includes administering by iv, sc, intrathecal, po, inhalation, topical, or other clinically acceptable administration method using a pharmaceutically acceptable excipient, carrier, or diluent selected from the group of pharmaceutical carriers, positive charge carriers, liposomes, lipid nanoparticles, protein carriers, polymers, nanoparticles, nanoemulsions, lipids, N-acetyl-galactosamine (GalNAc), lipophilic compounds or moieties, and lipoids.

[0168] In certain embodiments, the silencing method is used to determine the function or utility of a gene in a cell or organism.

[0169] In some embodiments, the gene or RNA targeted by the composition of the present invention is related to or involved in a disease, such as a human disease or an animal disease, a pathological condition, or an undesirable condition.In a further embodiment, the target gene or RNA is of pathogenic microorganism.In yet a further embodiment, the target gene or RNA is of viral origin.In another embodiment, the target gene or RNA is tumor-related.

[0170] In alternative embodiments, the gene or RNA targeted by the compositions of the invention is a gene or RNA associated with, or more specifically involved in, a cancer, an autoimmune disease, an inflammatory disease, a degenerative disease, an infectious disease, a proliferative disease, a metabolic disease, an immune-mediated disorder, an allergic disease, a dermatological disease, a malignant disease, a gastrointestinal disorder, a hepatic disorder, a respiratory disorder, a cardiovascular disorder, a dermatological disorder, a renal disorder, a rheumatic disorder, a neurological disorder, a psychiatric disorder, an endocrine disorder, or an aging-related disorder or disease.

[0171] 6.2 Treatment method The present invention also provides methods of treating or preventing various diseases or conditions, including those summarized for ASO and siRNA (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 include administering an effective amount of an sdDNA molecule to a subject in need thereof under conditions that allow for the desired gene inhibition described in the section immediately above.

[0172] In an exemplary embodiment, a pharmaceutical composition having an sdDNA molecule and a pharma- ceutically acceptable excipient, carrier, or diluent is administered in a therapeutically effective amount to a patient in need thereof to treat or prevent a disease or undesirable condition.

[0173] In some embodiments, the present invention can be used as cancer treatment or to prevent cancer.The composition of sdDNA can be used to silence or knock down genes involved in cell proliferation disorders or malignant diseases.Examples of these genes are k-Ras, β-catenin, Stat3.These oncogenes are active and associated with many human cancers.

[0174] The novel compositions of the present invention can also be used to treat or prevent ophthalmic diseases (e.g., age-related macular degeneration (AMD) and diabetic retinopathy (DR)); infectious diseases (e.g., HIV / AIDS, Hepatitis B virus (HBV), Hepatitis C virus (HCV), human papillomavirus (HPV), herpes simplex virus (HSV), RCV, cytomegalovirus (CMV), dengue fever, West Nile virus); respiratory diseases (e.g., respiratory syncytial virus (RSC), asthma, cystic fibrosis); neurological diseases (e.g., Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), spinal cord injury, Parkinson's disease, Alzheimer's disease, pain); cardiovascular diseases; metabolic disorders (e.g., hyperlipidemia, hypercholesterolemia, and diabetes); genetic disorders; and inflammatory conditions (e.g., inflammatory bowel disease (IBD), arthritis, rheumatoid diseases, autoimmune disorders), dermatological diseases.

[0175] In alternative embodiments, the method of administration is a route selected from the group of intravenous injection (iv), subcutaneous injection (sc), orally (po), intrathecal, inhalation, topical, and other localized administration. EXAMPLES

[0176] Examples are provided below to further illustrate different features of the present invention. The examples also illustrate useful methodologies for implementing the present invention. These examples do not limit the invention as claimed.

[0177] Methods and Materials cell culture HepaRG cells were grown in Williams' medium supplemented with 10% FBS, 10 mg / ml hydrocortisone, and 4 mg / ml human recombinant insulin.

[0178] Transfection of sdDNA into HepaRG cells 24 hours before transfection, HepaRG cells were seeded in 6-well plates (1x105 cells / 2mL / well). sdDNA was transfected with Lipofectamine® RNAiMAX (Thermo Fisher, USA) at a final concentration of 100pM as described in the manufacturing method. Briefly, sdDNA and RNAiMAX were incubated in serum-free OPTI-MEM (Thermo Fisher) for 20 minutes, and then added to cells together with culture medium.

[0179] quantitative PCR Cells transfected with the indicated sdDNA were harvested 48 hours post-transfection. RNA was isolated with TRIZOL and qRT-PCR was performed using TaqMan one-step RT-PCR reagents and the APOCIII assay for APOCIII mRNA detection, and gene GAPDH mRNA levels were used as an internal control.

[0180] Target sequence

[0181] In order to demonstrate the gene silencing effect of the sdDNA disclosed in the present invention, the sdDNA designed and constructed to target APOCIII gene (APOCIII mRNA) was used in the following examples. The target sequence is a segment from accession number NM_000040 that can be bound to the antisense strand of sdDNA or the corresponding single-stranded antisense oligonucleotide.

[0182] Example 1 Gene silencing activity of sdDNAs with various ISR motifs FIG. 1A shows the structure of the embodiment of sdDNA (sdDNA1-3) provided by the present invention, which has different ISR motifs and the ISR is located in both AS (antisense strand) and SS (sense strand) or only in AS, and the sequence of sdDNA (sdDNA1-3) designed to target APOCIII gene. Single-stranded antisense oligonucleotides having the same structure and sequence as the antisense strand of sdDNA (sdDNA1-3) were also used as corresponding single-stranded AS oligonucleotides (corresponding ASO) for comparison. The corresponding ASO (SEQ ID NO: 1) used here is ISIS304801, a typical ASO product optimized with the most advanced state-of-the-art ASO technology and know-how. The gene silencing activity targeting APOCIII by sdDNA1-3 and corresponding ASO was tested in HepaRG cells (results are shown in FIG. 1B).

[0183] In FIG. 1A, every letter "D" in the structures shown represents a DNA residue or a deoxyribonucleotide monomer; every letter "R" in the structures shown represents an RNA residue or a ribonucleotide monomer; every lowercase letter "a, c, g, t" in the sequences represents a DNA residue; every uppercase letter "A, C, G, U" in the sequences represents a 2'-MOE modified RNA residue, where every "U" is a 5-methyluridine 2'-MOE RNA residue; every "C" and "c" is 5-Me-C; every "*" in the structures shown represents a PS (phosphorothioate internucleoside linkage).

[0184] The results suggest that all the designed sdDNAs have high potency of gene silencing activity against APOCIII at very low concentrations (picomolar concentrations), and are significantly more potent and effective than the corresponding single-stranded antisense oligonucleotides (ASOs).

[0185] Example 2 Various lengths of sdDNA FIG. 2A shows some embodiments of sdDNA. In these sdDNAs, AS and SS are symmetric in length. Various sdDNA duplex lengths (8-36 bp) were designed (sdDNA_1-10 structure and sequence of sdDNA for targeting APOCIII gene shown in FIG. 2A). Single-stranded antisense oligonucleotides with identical structure and sequence to the antisense strand of sdDNA_1-10 were also made as the corresponding single-stranded AS of each sdDNA. The gene silencing activity of sdDNA_1-10 and each corresponding single-stranded ASO designed to target APOCIII was tested in HepaRG cells (results of sdDNA are shown in FIG. 2B and corresponding single-stranded AS are shown in FIG. 2C).

[0186] In FIG. 2A, all lower case "a, c, g, t", upper case "A, C, G, U" and "*" in the sequences shown represent the same as in FIG. 1A.

[0187] The results suggest that symmetric blunt-ended sdDNA with 10 bp has very strong gene silencing activity at very low concentrations (picomolar concentrations), and is significantly more potent and effective than the corresponding single-stranded ASO. Although the corresponding single-stranded ASOs here generally show low activity at picomolar concentration levels, they can show gene silencing activity at nanomolar concentration levels (about 10 nM to 30 nM), which is consistent with known oligonucleotides developed under the well-known current state-of-the-art ASO technology. Here, it is also a surprising discovery that single-stranded AS oligonucleotides having a much longer length than typical ASOs (having a length of approximately 16 nt to 20 nt) show stronger gene silencing activity than the typical ASOs. However, the sdDNA of the present invention can always show relatively better activity than the corresponding single-stranded AS oligonucleotides, including the known typical well-optimized ASOs (e.g., SEQ ID NO: 1) by the state-of-the-art ASO technology.

[0188] Example 3 Symmetric sdDNA of various lengths Figure 3A shows different structural designs of sdDNA embodiments. In these sdDNAs, AS and SS are symmetric in length. Furthermore, various lengths of AS and SS duplexes, from 8 to 14 bp, were designed and fabricated (sdDNA_1-3 structures and sequences of sdDNA for targeting APOC gene, shown in Figure 3A). The gene silencing activity of sdDNA_1-3 designed to target APOCIII was tested in HepaRG cells (sdDNA results shown in Figure 2B).

[0189] In FIG. 3A, all lowercase "a, c, g, t" and "*" in the sequences shown represent the same as in FIG. 1A, and all underlined capital letters " A , C , G , U " represents an LNA modified RNA residue, in which case all " U " is a 5-methyluridine LNA RNA residue, and all " C " is a 5-Me-C LNA RNA residue.

[0190] The results suggest that all the designed sdDNAs have extremely potent gene silencing activity at very low concentrations (picomolar concentrations).

[0191] Example 4 sdDNA with various modified motifs in SS FIG. 4A shows different structural designs of embodiments of sdDNA with different PS modification motifs in SS. Specifically, in these sdDNAs, AS was kept constant and SS was kept the same except for the internucleoside linkages: specifically, sdDNA_SS:PS has an all-SS where each internucleoside linkage is PS-modified, sdDNA_SS:PO has an all-SS where each internucleoside linkage is a natural PO internucleoside linkage, and sdDNA_SS:PS / PO has a mixed SS where only some, but not all, of the internucleoside linkages of the linked deoxyribonucleoside monomers are PS-modified internucleoside linkages. Single-stranded antisense oligonucleotides with the same structure and sequence as the antisense strands of sdDNA (sdDNA1-3) were also used as the corresponding single-stranded AS oligonucleotides (corresponding ASOs) for comparison. The corresponding ASO (SEQ ID NO: 1) used here is ISIS304801, a typical ASO product optimized with the most advanced state-of-the-art ASO technology and know-how. The gene silencing activity of the designed sdDNA (PS, PO, PS / PO) and the corresponding ASO targeting APOCIII was tested in HepaRG cells (results shown in Figure 4B).

[0192] In FIG. 4A, all lower case "a, c, g, t", upper case "A, C, G, U" and "*" in the sequences shown represent the same as in FIG. 1A.

[0193] All the designed sdDNAs with different PS modification motifs in SS show extremely strong gene silencing activity against APOCIII at very low concentration (picomolar concentration), which is significantly more potent and effective than the corresponding ASO. In other words, various modification motifs in the internucleoside linkages of linked deoxyribonucleoside monomers / DNA residues can maintain the advantage of significant improvement of gene silencing activity by the gene silencing technology based on sdDNA provided in the present invention, at least compared with traditional ASO technology. The sdDNAs with SS partially or completely modified at PS internucleoside linkages showed strong gene silencing activity that is at least as good as that of sdDNAs with completely natural PO internucleoside linkages. Moreover, it is well known in the art that PS modification is required for in vivo application.

[0194] These results also suggest that SS is removed or degraded from sdDNA duplexes by an unknown mechanism other than typical DNases, because sdDNA with a fully PS-modified SS is extremely potent or at least as potent as sdDNA with an unmodified SS. Furthermore, it is also expected that using at least one modification of the linked deoxyribonucleoside monomer / DNA residue within the SS (e.g., PS-modified internucleoside linkages) can help design sdDNA with improved pharmaceutical properties for in vivo applications, including better stability and tissue distribution.

[0195] Example 5 sdDNA with various ISR motifs located at various positions in the AS Figure 5A shows a further series of sdDNAs with different structural designs. In these sdDNAs, the sense strand was kept constant, but the position and total number of ribonucleotide monomers of the ISR in the AS were varied (ISR_0-5, structures and sequences shown in Figure 5A). The various ISR motifs in the antisense strand in Figure 5A show that each ISR has a small number of ribonucleotide monomers, such as one or two, and that each ISR is spaced apart by at least one intervening deoxyribonucleotide monomer. In Figure 5A, all lowercase letters "a, c, g, t", capital letters "A, C, G, U" and "*" in the sequences shown represent the same as in Figure 1A. The gene silencing activity of ISR_0-5 designed to target APOCIII was tested in HepaRG cells (results shown in Figure 5B).

[0196] All the designed sdDNAs have extremely strong gene silencing activity at very low concentration (picomolar concentration level).The results further suggest that at least one ISR in AS, which has various numbers of ribonucleotide monomers and is arranged at various positions of AS, can enhance or enable the extremely strong gene silencing activity of the sdDNAs provided in the present invention.

[0197] Example 6 sdDNA with mismatches in AS Figure 6A shows a further series of sdDNAs with different structural designs. In these sdDNAs, the antisense strand was designed to contain at least one mismatch when hybridized to the target RNA (Mis1-2, structures and sequences shown in Figure 6A), and an antisense strand without mismatch (Mis0) was designed as a comparison. In Figure 6A, all lowercase letters "a, c, g, t", capital letters "A, C, G, U" and "*" in the sequences shown represent the same as those in Figure 1A. The gene silencing activity of Mis_0-3 designed to target APOCIII was tested in HepaRG cells (results shown in Figure 6B).

[0198] All designed sdDNAs have extremely strong gene silencing activity at very low concentration (picomolar concentration level).The results further suggest that the antisense strand of the sdDNA provided in the present invention can have at least two (at least 10% of the targeting region) mismatches while maintaining the gene silencing activity of the sdDNA provided in the present invention.Some mismatches or multiple mismatches at certain positions in AS may reduce the gene silencing activity of sdDNA.

[0199] Example 7 sdDNA with SS longer than AS Figure 7A shows different structural designs of a further series of sdDNAs with longer SS than AS. In these sdDNAs, the antisense strand was kept constant, but the length of the sense strand was changed (sdDNA_1-2, structures and sequences shown in Figure 7A). In Figure 7A, all lowercase letters "a, c, g, t", capital letters "A, C, G, U" and "*" in the sequences shown represent the same as those in Figure 1A. Single-stranded antisense oligonucleotides with identical structures and sequences to the antisense strands of sdDNAs (sdDNA_1-2) were also used as corresponding single-stranded AS oligonucleotides (corresponding ASOs) for comparison. The gene silencing activity targeting APOCIII by the designed sdDNA1-2 and corresponding ASOs was tested in HepaRG cells (results shown in Figure 7B).

[0200] All designed sdDNAs have extremely strong gene silencing activity at very low concentration (picomolar level), and are significantly more potent and effective than corresponding ASOs.The results suggest that the sense strand of sdDNA can be longer than the antisense strand.As shown in this example, SS can be longer than AS by at least 16 monomers.

[0201] Equivalent The representative examples are intended to serve as illustrations of the invention, and are not intended to, and should not be construed as, limiting the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the complete contents of this document, including the examples contained herein and references to the scientific and patent literature. The examples contain important additional information, exemplification and guidance that may be applicable to the practice of the invention in its various embodiments and their equivalents.

[0202] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.Although any method and material similar or equivalent to those described herein can be used in carrying out or testing this disclosure, preferred methods and materials are now described.The methods described herein can be carried out in any order that is logically possible, in addition to the specific order disclosed.

[0203] Incorporation by Reference References and citations to other documents, such as patents, patent applications, patent publications, periodicals, books, academic papers, web content, etc., are made in this disclosure. All such documents are hereby incorporated by reference in their entirety for all purposes. Any material or portion thereof that is stated to be incorporated by reference in this specification, but which conflicts with existing definitions, descriptions or other disclosed materials clearly indicated in this specification, is incorporated only to the extent that no conflict occurs between the incorporated material and the disclosed material. In case of conflict, the conflict should be resolved in favor of the present disclosure as the preferred disclosure. References [ka] [ka] [ka]

Claims

1. A short double-stranded DNA (sdDNA) molecule comprising a first strand and a second strand, each containing linked nucleotide monomers, wherein the first strand has a length 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, and is substantially complementary to a segment of the target RNA that is the target, and forms a target region, wherein the second strand is equal in length to the first strand or is longer than the first strand by a number of nucleotide monomers selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 monomers, and is substantially complementary to the first strand, and forms at least one double-stranded region with the first strand, wherein the sdDNA molecule contains at least one ribonucleotide monomer interspersed segment (ISR), and each ISR independently consists of one ribonucleotide monomer or consists of two, three, four, five, six or more than six consecutive ribonucleotide monomers, wherein the first strand contains at least one ISR, wherein the second strand (a) contains at least one ISR, (b) consists of deoxynucleotide monomers, or (c) contains at least one nucleotide monomer that is a modified nucleotide or a nucleotide analog, sdDNA molecule.

2. The sdDNA molecule according to claim 1, wherein at least one of the ISRs in the first strand consists of two, three, four, five, six or more than six consecutive ribonucleotide monomers.

3. (a) The first strand is at least 70%, 80%, 85%, 90%, 95% complementary or completely complementary to the segment of the target RNA that is the target, and (b) the double-stranded region has base pair complementarity at a percentage selected from the group consisting of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, and any number therebetween, The sdDNA molecule according to claim 1.

4. The second strand is either fully complementary to the first strand or contains one, two, three or more mismatches upon formation of the complementary double strand, and the mismatch monomer in the second strand has a nucleobase selected from the group consisting of A, G, C and T, the sdDNA molecule according to claim 1.

5. The double-stranded region consists of a number of base pairs 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 base pairs, the sdDNA molecule according to claim 1.

6. The two ends of the second strand are (a) both ends are blunt ends, (b) 3'-overhang and 5'-overhang, (c) 3'-overhang and 5'-blunt end, (d) 3'-blunt end and 5'-overhang, (e) 3'-overhang and 5'-recessed end, and (f) 5'-overhang and 3'-recessed end, and are configured to be selected from the group consisting of, the 3'-overhang or the 5'-overhang having at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide monomers, the sdDNA molecule according to claim 1.

7. (a) 50 percent or more of the nucleoside monomers within the RNA targeting motif are deoxyribonucleoside monomers, or (b) at least 50% of the nucleotides within the first strand in the RNA targeting portion of the double-stranded region are deoxyribonucleotide monomers, or (c) at least half of the nucleobases within at least one strand of the double-stranded region are deoxyribonucleotide monomers, or (d) 50 percent or more of the nucleobases within one strand of the double-stranded region of the sdDNA molecule contain deoxyribonucleoside monomers, the sdDNA molecule according to claim 1.

8. At least one nucleotide monomer is a modified nucleotide or nucleotide analog, and the modified nucleotide or nucleotide analog is (a) A backbone-modified nucleotide, wherein the backbone-modified nucleotide has a modification of the internucleoside linkage, and the internucleoside linkage is modified to include at least one of a nitrogen or sulfur heteroatom, the backbone-modified nucleotide. (b) A modified sugar moiety, and / or (c) A modified nucleobase The sdDNA molecule according to claim 1, comprising.

9. In (a), (a) The modified internucleoside linkage is selected from the group consisting of phosphorothioate (P=S) groups, phosphotriesters, methylphosphonates, and phosphoramidates, or (b) The first strand and / or the second strand comprises at least one modified internucleoside linkage, and the modified internucleoside linkage is a phosphorothioate internucleoside linkage, or (c) The second strand comprises at least one modified internucleoside linkage in the linked deoxyribonucleotide monomer region, and the modified internucleoside linkage is a phosphorothioate internucleoside linkage, or (d) Each internucleoside linkage of the first strand and / or the second strand is a phosphorothioate internucleoside linkage. The sdDNA molecule according to claim 8.

10. In (b), (a) The 2'-position of the modified sugar moiety is replaced by a group selected from the group consisting of OR, R, halo, SH, SR, NH ２ , NHR, NR ２ , and CN, wherein each R is independently C １ -C ６ alkyl, alkenyl or alkynyl, and halo is F, Cl, Br or I, or (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-C1-C10 alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-O-N(Rm)(Rn), O-CH2-C(=O)-N(Rm)(Rn), and O-CH2-C(=O)-N(R1)-(CH2)2-N(Rm)(Rn), wherein each of Rl, Rm, and Rn is independently H or substituted or unsubstituted C1-C10 alkyl, or (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) the modified sugar moiety is replaced by a bicyclic sugar selected from the group consisting of 4'-(CH₂)-O-2'(LNA); 4'-(CH₂)-S-2; 4'-(CH₂)₂-O-2'(ENA); 4'-CH(CH₃)-O-2'(cEt) and 4'-CH(CH₂OCH₃)-O-2', 4'-C(CH₃)(CH₃)-O-2', 4'-CH₂-N(OCH₃)-2', 4'-CH₂-O-N(CH₃)-2', 4'-CH₂-N(R)-O-2' (wherein R is H, C₁-C₁₂ alkyl, or a protecting group), 4'-CH₂-C(H)(CH₃)-2', and 4'-CH₂-C-(=CH₂)-2', or (e) the modified sugar moiety is selected from the group consisting of 2'-O-methoxyethyl modified sugar (MOE), 4'-(CH₂)-O-2' bicyclic sugar (LNA), 2'-deoxy-2'-fluoroarabinose (FANA), and methyl(methyleneoxy)(4'-CH(CH₃)-O-2) bicyclic sugar (cEt), or (f) the modified sugar moiety of the deoxynucleotide monomer is 2'-deoxy-2'-fluoroarabinose (FANA). The sdDNA molecule according to claim 8.

11. In (c), (a) the modified nucleobase is 5-methylcytosine (5-Me-C), inosine base, tritylated base, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine and 2-thiocytosine, 1-methyl-pseudo-uracil, 5-halouracil and cytosine; 5-propynyl (-C≡C-CH ３ ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases; 6-azauracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, in particular, 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, and 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine, or is selected from the group consisting of (b) the modified nucleobase is 5-methylcytosine, or (c) each cytosine base is 5-methylcytosine. The sdDNA molecule according to claim 8.

12. The sdDNA molecule according to claim 1, which is more potent or effective than the corresponding single-stranded antisense oligonucleotide or the corresponding siRNA in silencing the target RNA.

13. (a) a cell, (b) a eukaryotic cell, or (c) a mammalian cell The sdDNA molecule according to claim 1, which is used to modulate gene expression or function.

14. (a) the target RNA is either mRNA or non-coding RNA, and such RNA encodes a protein involved in a disease or condition, or controls a part of a biological pathway involved in a disease or condition, or (b) the target RNA is (i) the mRNA of a gene involved in a human or animal disease or disorder, (ii) the mRNA of a gene of a pathogenic microorganism, (iii) viral RNA, and RNAs involved in diseases selected from the group consisting of autoimmune diseases, inflammatory diseases, degenerative diseases, infectious diseases, proliferative diseases, metabolic diseases, immune-mediated disorders, allergic diseases, dermatological diseases, malignant diseases, gastrointestinal disorders, respiratory disorders, cardiovascular disorders, renal disorders, rheumatism-like disorders, neurological disorders, endocrine disorders, and aging-related disorders selected from the group consisting of the sdDNA molecule according to claim 1

15. The sdDNA molecule according to claim 1, wherein the first strand and / or the second strand is conjugated to a ligand or a moiety, and the ligand or moiety is selected from the group consisting of peptides, antibodies, polymers, polysaccharides, lipids, hydrophobic moieties or molecules, cationic moieties or molecules, lipophilic compounds or moieties, oligonucleotides, cholesterol, GalNAc, and aptamers

16. A pharmaceutical composition comprising the sdDNA molecule according to any one of claims 1 to 15 as an active agent and a pharmaceutically acceptable excipient, carrier or diluent

17. A pharmaceutical composition for treating or preventing a disease or condition in a subject in need thereof, or for modulating gene expression or gene function in eukaryotic cells, comprising the sdDNA molecule according to any one of claims 1 to 15

18. The pharmaceutical composition according to claim 17, wherein the disease or condition is selected from the group consisting of cancer, autoimmune diseases, inflammatory diseases, degenerative diseases, infectious diseases, proliferative diseases, metabolic diseases, immune-mediated disorders, allergic diseases, dermatological diseases, malignant diseases, gastrointestinal disorders, liver disorders, respiratory disorders, cardiovascular disorders, dermatological disorders, renal disorders, rheumatism-like disorders, neurological disorders, mental disorders, endocrine disorders, and aging-related disorders or diseases

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