RNA degradation via the lysosomal pathway

Abstract

This disclosure provides oligonucleotides, methods, and compositions for degrading RNA via the lysosomal pathway. It also envisions oligonucleotides, methods, and compositions for treating, preventing, or improving diseases, disorders, and conditions associated with EXOC2, Ku80, and Task1 in subjects requiring such treatment.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 532,208, filed on 11 August 2023. The entire contents of the patent application referenced above are incorporated herein by reference. [Background technology]

[0002] RNA knockdown-based approaches have emerged as powerful tools for manipulating gene expression and elucidating gene function (Fire et al., 1998; Hannon, 2002). Utilizing small RNA molecules such as small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), these methods offer the potential for selectively silencing gene expression with high precision and specificity (Elbashir et al., 2001; Brummelkamp et al., 2002). Despite their potential, these approaches face challenges, particularly regarding efficacy and target specificity (Jackson & Linsley, 2010; Boudreau et al., 2011).

[0003] One of the major constraints encountered in RNA knockdown methods is related to the intracellular fate of RNA molecules. Cellular uptake and subsequent processing are complex processes that can significantly affect knockdown efficiency (Juliano, 2016). Furthermore, suboptimal endosomal escape and premature degradation in the lysosomal compartment have been identified as major obstacles hindering the effectiveness of these approaches (Gilleron et al., 2013; Daka et al., 2020).

[0004] Therefore, there is a need for more efficient RNA knockdown methods. [Overview of the project]

[0005] This disclosure provides oligonucleotides, methods, and compositions for degrading RNA via the lysosomal pathway.

[0006] In one embodiment, the disclosure provides an oligonucleotide comprising a 5' end, a 3' end, and complementarity to a target polynucleotide, or comprising a poly-G sequence ligated to the 5' end and / or 3' end of the oligonucleotide, wherein the poly-G sequence lacks complementarity to the target polynucleotide.

[0007] In some embodiments, the poly-G sequence contains 2 to 30 G nucleotides.

[0008] In some embodiments, the poly-G sequence contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 5 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 6 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 7 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 8 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 9 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 10 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 11 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 12 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 13 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 14 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 15 G nucleotides.

[0009] In some embodiments, the poly-G sequence is ligated to the 3' end of the oligonucleotide of the disclosure. In other embodiments, the poly-G sequence is ligated to the 5' end of the oligonucleotide of the disclosure. In other embodiments, the poly-G sequence is ligated to both the 5' and 3' ends of the oligonucleotide of the disclosure.

[0010] In some embodiments, the poly-G sequence is single-stranded.

[0011] In some embodiments, the poly-G sequence is bound to a protein on the surface of a lysosome. In some embodiments, the poly-G sequence is bound to a lysosome-associated membrane glycoprotein (LAMP). In some embodiments, the LAMP is LAMP2C.

[0012] In some embodiments, the poly-G sequence contains consecutive G nucleotides. In some embodiments, the poly-G sequence contains or consists of 5 to 15 consecutive G nucleotides. In some embodiments, the poly-G sequence contains one or more non-G nucleotides (e.g., A, T, U, or C) within the poly-G sequence. In some embodiments, the oligonucleotide contains two or more poly-G sequences having one or more non-G nucleotides (e.g., A, T, U, or C) between them.

[0013] In some embodiments, the poly-G sequence includes G nucleotides that are consecutive, discontinuous, or a combination thereof.

[0014] In some embodiments, the poly-G sequence includes or consists of (dG)(dG)(dG)(dG)(dG). In some embodiments, the poly-G sequence includes or consists of (dG)(dG)(dG)(dG)(dG)(dG). In some embodiments, the poly-G sequence includes or consists of (dG)(dG)(dG)(dG)(dG)(dG)(dG). In some embodiments, the poly-G sequence includes or consists of (dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG). In some embodiments, the poly-G sequence includes or consists of (dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG). In some embodiments, the polyG sequence includes or consists of (dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG). In some embodiments, the polyG sequence includes or consists of (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dN)(dG), where each dN corresponds individually to a deoxyribonucleotide of dA, dT, or dC. In some embodiments, the polyG sequence includes or consists of (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dN)(dG)(dG), where each dN corresponds individually to a deoxyribonucleotide of dA, dT, or dC. In some embodiments, the polyG sequence contains or consists of (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dN)(dG)(dG), where each dN corresponds individually to a deoxyribonucleotide of dA, dT, or dC.

[0015] In some embodiments, the oligonucleotides are approximately 10 to 35 nucleotides in length.

[0016] In some embodiments, the oligonucleotides have a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 nucleotides.

[0017] In some embodiments, the oligonucleotide and / or poly-G sequence comprises one or more modified nucleotides.

[0018] In some embodiments, one or more modified nucleotides each independently include modifications of a ribose group, a phosphate group, a nucleic acid base, or a combination thereof.

[0019] In some embodiments, each modification of the ribose group includes 2'-O-methyl, 2'-fluoro, 2'-deoxy, 2'-O-(2-methoxyethyl)(MOE), 2'-O-alkyl, 2'-O-alkoxy, 2'-O-alkylamino, 2'-NH2, a restricted nucleotide, or a combination thereof.

[0020] In some embodiments, the restricted nucleotides include lock nucleic acids (LNA), ethyl-restricted nucleotides, 2'-(S)-restricted ethyl (S-cEt) nucleotides, restricted MOEs, 2'-O,4'-C-aminomethylene-bridged nucleic acids (2',4'-BNANC), alpha-L-lock nucleic acids, tricyclo-DNA, or combinations thereof.

[0021] In some embodiments, the modification of the ribose group includes a 2'-O-(2-methoxyethyl)(MOE) modification.

[0022] In some embodiments, the nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and / or 10 from the 5' and / or 3' ends of the oligonucleotide include a 2'-O-(2-methoxyethyl)(MOE) modification.

[0023] In some embodiments, all nucleotides of the oligonucleotide and / or poly-G sequence include a 2'-O-(2-methoxyethyl)(MOE) modification.

[0024] In some embodiments, the modification of the ribose group includes tricyclo-DNA modification.

[0025] In some embodiments, all nucleotides of the oligonucleotide and / or poly-G sequence include tricyclo-DNA modifications.

[0026] In some embodiments, the modification of the ribose group includes a 2'-deoxy modification.

[0027] In some embodiments, each modification of the phosphate group includes phosphorothioates, phosphonoacetic acid (PACE), thiophosphonoacetic acid (thioPACE), amides, triazoles, phosphonates, phosphotryesters, or combinations thereof.

[0028] In some embodiments, the modification of the phosphate group is a phosphorothioate.

[0029] In some embodiments, all nucleotides in the oligonucleotide and / or poly-G sequence contain phosphorothioates.

[0030] In some embodiments, the oligonucleotide and / or poly-G sequence includes at least one phosphodiester nucleotide linkage.

[0031] In some embodiments, all internucleotide links in the oligonucleotide and / or poly-G sequence are phosphodiester nucleotide links.

[0032] In some embodiments, each modification of the nucleic acid base includes 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, halogenated aromatic groups, or combinations thereof.

[0033] In some embodiments, the modification of the nucleic acid base group includes 5-methylcytosine modification.

[0034] In some embodiments, the oligonucleotide comprises a mixture of modified nucleotides.

[0035] In some embodiments, the functionalized moiety is ligated to the 5' or 3' end of the oligonucleotide.

[0036] In some embodiments, the functionalized moiety includes an N-acetylgalactosamine (GalNAc) moiety and / or a hydrophobic moiety.

[0037] In some embodiments, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and mixtures thereof.

[0038] In some embodiments, the steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA).

[0039] In some embodiments, the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanic acid (DCA).

[0040] In some embodiments, the oligonucleotide is expressed by formula Including ABC, in the formula, A contains approximately 0 to 8 modified nucleotides. B contains approximately 6 to 18 deoxyribonucleic acid (DNA) nucleotides and / or DNA-like nucleotides. C contains approximately 0 to 8 modified nucleotides. The total length of an antisense oligonucleotide is approximately 10 to 30 nucleotides.

[0041] In some embodiments, A comprises about 2 to about 6 modified nucleotides, B comprises about 6 to about 12 DNA nucleotides and / or DNA-like nucleotides, and C comprises about 2 to about 6 modified nucleotides.

[0042] In some embodiments, A comprises about 5 modified nucleotides, B comprises about 10 DNA nucleotides and / or DNA-like nucleotides, and C comprises about 5 modified nucleotides.

[0043] In some embodiments, A comprises about 2 to about 6 2'-O-(2-methoxyethyl) (MOE) modified nucleotides, B comprises about 6 to about 12 DNA-like nucleotides, and C comprises about 2 to about 6 2'-O-(2-methoxyethyl) (MOE) modified nucleotides.

[0044] In some embodiments, A comprises about 5 2'-O-(2-methoxyethyl) (MOE) modified nucleotides, B comprises about 10 DNA-like nucleotides, and C comprises about 5 2'-O-(2-methoxyethyl) (MOE) modified nucleotides.

[0045] In some embodiments, the oligonucleotide comprises a nucleic acid sequence having at least 90% sequence identity to any one of the nucleic acid sequences in Table 1.

[0046] In some embodiments, the oligonucleotide is X S , S , S , S , S , S , S , X , S , X , X , S , S , S , S , S , X , S , S , X , S , X X S X S X S X S X S X S X S X S X S X S X S X S X S X S X S X S X S X S XThe formula includes the array modification pattern, s represents the linkage between phosphorothioate nucleosides, X It contains adenosine, guanosine, cytidine, thymine, or uracil. X It includes a 2'-O-(2-methoxyethyl) modification, X contains adenosine, guanosine, cytidine, thymine, or uracil, and X contains a 2'-deoxy modification.

[0047] In some embodiments, the oligonucleotide is X S X S X S X S X S X S X S X S X S X S X S X S X S X S X S X S X S X The formula includes the array modification pattern, s represents the linkage between phosphorothioate nucleosides, X It contains adenosine, guanosine, cytidine, thymine, or uracil. X This includes a 2'-O-(2-methoxyethyl) modification.

[0048] In some embodiments, the oligonucleotide is X S X SX S X S X S X S X S X S X S X S X S X S X S X S X S X S X S X S X S X The formula includes the array modification pattern, s represents the linkage between phosphorothioate nucleosides, X It contains adenosine, guanosine, cytidine, thymine, or uracil. X This includes a 2'-O-(2-methoxyethyl) modification.

[0049] In some embodiments, the target polynucleotide is mammalian or viral mRNA. In some embodiments, the target polynucleotide is an intronic or exonic region of mRNA.

[0050] In some embodiments, the target is selected from the group consisting of the EXOC2 gene, the Ku80 gene, and the Task1 gene.

[0051] In some embodiments, the oligonucleotide inhibits the expression of the EXOC2 gene, Ku80 gene, or Task1 gene by at least about 50%.

[0052] In some embodiments, the target is SOD1.

[0053] In some embodiments, the oligonucleotide comprises the sequence (eC)#(eA)(eG)#(eG)(eA)#(dT)#(dA)#(d5C)#(dA)#(dT)#(dT)#(dT)#(d5C)#(dT)#(dA)#(eC)(eA)#(eG)(eC)#(eU)(polyG), where (#) indicates a phosphorothioate linkage, (e) indicates a 2'MOE modification, (d) indicates a deoxyribonucleotide, (dN) indicates a deoxyribonucleotide of A, T, or C, (d5C) indicates 5-methylcytosine, and (polyG) indicates 2 to 30 G nucleotides.

[0054] In some embodiments, the oligonucleotide has the sequence (eC)#(eA)(eG)#(eG)(eA)#(dT)#(dA)#(d5C)#(dA)#(dT)#(dT)#(dT)#(d5C)#(dT)#(dA)#(eC)(eA)#(eG)(eC)#(eU)(dG) x The formula includes, where (#) indicates a phosphorothioate linkage, (e) indicates a 2'MOE modification, (d) indicates a deoxyribonucleotide, (d5C) indicates 5-methylcytosine, and x indicates an integer between 5 and 10.

[0055] In some embodiments, oligonucleotides are in the sequence (eC)#(eA)(eG)#(eG)(eA)#(dT)#(dA)#(d5C)#(dA)#(dT)#(dT)#(dT)#(d5C)#(dT)#(dA)#(eC)(eA)#(eG)(eC)#(eU)(dGdGdGdN) x The formula includes, where (#) indicates a phosphorothioate linkage, (e) indicates a 2'MOE modification, (d) indicates a deoxyribonucleotide, (d5C) indicates 5-methylcytosine, (dN) indicates a deoxyribonucleotide of A, T, or C, and x indicates an integer from 2 to 10.

[0056] In some embodiments, the target is PCSK9.

[0057] In some embodiments, the oligonucleotide is selected from the group consisting of antisense oligonucleotides (ASOs), gapmers, siRNAs, miRNAs, shRNAs, CRISPR guides, DNAs, antisense mixmers, miRNA inhibitors, splice-switching oligonucleotides (SSOs), phosphorodiamidate morpholino oligomers (PMOs), and peptide nucleic acids (PNAs).

[0058] In some embodiments, the oligonucleotide is double-stranded RNA (dsRNA).

[0059] In some embodiments, the dsRNA includes an antisense strand complementary to the target polynucleotide. In some embodiments, the antisense strand is approximately 10–35 nucleotides long. In some embodiments, the antisense strand is 18, 19, 20, 21, 22, or 23 nucleotides long. In some embodiments, the dsRNA includes a sense strand complementary to at least a portion of the antisense strand. In some embodiments, the sense strand is approximately 10–35 nucleotides long. In some embodiments, the sense strand is 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides long.

[0060] In some embodiments, the poly-G sequence is ligated to the 5' and / or 3' ends of the antisense strand and / or sense strand.

[0061] In some embodiments, the target is PCSK9.

[0062] In some embodiments, the dsRNA consists of an antisense strand of V(mA)#(fC)#(mA)(fA)(fA)(mG)(fC)(mA)(fA)(mA)(mA)(mC)(fA)(mG)(fG)(mU)(fC)(mU)(mA)(mG)#(mA)#(mA) and (mC)#(mU)#(mA)(mG)(mA)(mC)(fC)(mU)(fG)(mU)( The formula includes the sense chain of dT)(mU)(mU)(mG)(mC)(mU)(mU)(mU)(mU)(mG)(mU)GalNac, where (#) indicates phosphorothioate linkage, (mN) indicates 2'-OMe modification, (fN) indicates 2'-fluoro modification, V indicates 5'-vinyl phosphate, and GalNac indicates N-acetylgalactosamine (GalNAc) conjugate.

[0063] In some embodiments, the dsRNA comprises an antisense strand of V(mA)#(fC)#(mA)(fA)(fA)(mG)(fC)(mA)(fA)(mA)(mA)(mC)(fA)(mG)(fG)(mU)(fC)(mU)(mA)(mG)#(mA)#(mA)(polyG), where (#) indicates a phosphorothioate linkage, (mN) indicates a 2'-OMe modification, (fN) indicates a 2'-fluoro modification, V indicates a 5'-vinyl phosphate, and (polyG) indicates 2 to 30 G nucleotides.

[0064] In some embodiments, dsRNA is V(mA)#(fC)#(mA)(fA)(fA)(mG)(fC)(mA)(fA)(mA)(mA)(mC)(fA)(mG)(fG)(mU)(fC)(mU)(mA)(mG)#(mA)#(mA)(dG) x The formula includes an antisense chain, where (#) indicates a phosphorothioate linkage, (mN) indicates a 2'-OMe modification, (fN) indicates a 2'-fluoro modification, V indicates a 5'-vinyl phosphate, and x is an integer between 5 and 10.

[0065] In another aspect, the disclosure provides a pharmaceutical composition for inhibiting gene expression in an organism, comprising an oligonucleotide or dsRNA and a pharmaceutically acceptable carrier.

[0066] In some embodiments, the gene is selected from the group consisting of the EXOC2 gene, the Ku80 gene, and the Task1 gene.

[0067] In some embodiments, oligonucleotides or dsRNAs inhibit the expression of the EXOC2 gene, Ku80 gene, or Task1 gene by at least about 50%.

[0068] In some embodiments, oligonucleotides or dsRNAs inhibit the expression of the EXOC2 gene, Ku80 gene, or Task1 gene by at least about 80%.

[0069] In another aspect, the disclosure provides a vector comprising a regulatory sequence operably ligated to a nucleotide sequence encoding an oligonucleotide or dsRNA.

[0070] In some embodiments, oligonucleotides or dsRNAs inhibit gene expression by at least 30%.

[0071] In some embodiments, oligonucleotides or dsRNAs inhibit gene expression by at least about 50%.

[0072] In some embodiments, oligonucleotides or dsRNAs inhibit gene expression by at least about 80%.

[0073] In some embodiments, the gene is selected from the group consisting of the EXOC2 gene, the Ku80 gene, and the Task1 gene.

[0074] In another aspect, the disclosure provides recombinant adeno-associated virus (rAAV) comprising a vector and an AAV capsid.

[0075] In another embodiment, the present disclosure provides cells containing a vector or rAAV.

[0076] In another aspect, the present disclosure provides a method for inhibiting gene expression in cells, the method is (a) A step of introducing oligonucleotides, dsRNA, vectors, or rAAV into cells, (b) The step of maintaining the cells generated in step (a) for a sufficient time to achieve degradation of the gene mRNA transcript, thereby inhibiting gene expression in the cells.

[0077] In some embodiments, the poly-G sequence induces degradation from lysosomes.

[0078] In some embodiments, the gene is selected from the group consisting of the EXOC2 gene, the Ku80 gene, and the Task1 gene.

[0079] In some embodiments, oligonucleotides, dsRNAs, vectors, or rAAVs inhibit the expression of the EXOC2 gene, Ku80 gene, or Task1 gene by at least about 50%.

[0080] In some embodiments, oligonucleotides, dsRNAs, vectors, or rAAVs inhibit the expression of the EXOC2 gene, Ku80 gene, or Task1 gene by at least about 80%.

[0081] In some embodiments, oligonucleotides, dsRNAs, vectors, or rAAVs are administered by intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof.

[0082] In another aspect, the disclosure provides a method for treating or managing a gene-related disease, the method comprising the step of administering a therapeutically effective amount of oligonucleotide, dsRNA, vector, or rAAV to a patient in need of such treatment.

[0083] In some embodiments, the gene is selected from the group consisting of the EXOC2 gene, the Ku80 gene, and the Task1 gene.

[0084] In some embodiments, oligonucleotides, dsRNAs, vectors, or rAAVs inhibit the expression of the EXOC2 gene, Ku80 gene, or Task1 gene by at least about 50%.

[0085] In some embodiments, oligonucleotides, dsRNAs, vectors, or rAAVs inhibit the expression of the EXOC2 gene, Ku80 gene, or Task1 gene by at least about 80%.

[0086] In another aspect, the Disclosure provides a method for degrading a target polynucleotide in the lysosomes of a cell, the method comprising the steps of introducing an oligonucleotide, a dsRNA, a vector, or an rAAV as described herein into a cell, and maintaining the cell for a sufficient time to degrade the target polynucleotide in the lysosomes of the cell.

[0087] In some embodiments, methods for degrading target polynucleotides in cellular lysosomes occur in vivo, ex vivo, or in vitro.

[0088] In some embodiments, the target polynucleotide is mammalian or viral mRNA. In some embodiments, the target polynucleotide is an intronic or exonic region of mRNA.

[0089] In some embodiments, the target is selected from the group consisting of the EXOC2 gene, the Ku80 gene, and the Task1 gene.

[0090] In some embodiments, the oligonucleotide inhibits the expression of the EXOC2 gene, Ku80 gene, or Task1 gene by at least about 50%.

[0091] In another aspect, the present disclosure provides a method for treating or managing amyotrophic lateral sclerosis (ALS) in a patient, the method comprising the step of administering to the patient a therapeutically effective amount of an oligonucleotide complementary to SOD1 as described herein.

[0092] In another aspect, the disclosure provides a method for treating or managing primary hyperlipidemia in a patient, the method comprising the step of administering a therapeutically effective dose of a PCSK9-targeting dsRNA as described herein to the patient.

[0093] In some embodiments, primary hyperlipidemia is heterozygous familial hypercholesterolemia (HeFH).

[0094] In another aspect, the present disclosure provides a method for reducing low-density lipoprotein cholesterol (LDL-C) in a patient, the method comprising the step of administering a therapeutically effective dose of a PCSK9-targeting dsRNA as described herein to the patient.

[0095] The above and other features and advantages of this disclosure will be more fully understood from the detailed description of the exemplary embodiments in conjunction with the accompanying drawings. The patent or application file includes at least one drawing drawn in color. A copy of this patent or patent application publication having color drawings(s) will be provided by the Patent Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0096] [Figure 1] A schematic diagram of RNase H and lysosome-based knockdown approaches for knocking down target RNA is shown. [Figure 2]A schematic diagram of an exemplary Lyso-ASO capable of targeting lysosomes is shown. The gapmer ASO contains a 5' / 3' terminal 2'MOE modification with an internal DNA nucleotide. Each nucleotide linkage in the ASO is a phosphorothioate modification. The lyso sequence is unmodified. [Figure 3] This shows the relative EXOC2 mRNA levels measured by qRT-PCT analysis for gapmers with and without poly(G), poly(A), or poly(C) ligands. N=7–9. Values ​​are mean ± SEM. According to two-tailed Student's t-tests, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 4] The relative EXOC2 mRNA levels measured by qRT-PCT analysis are shown for gapmers with and without poly-G ligands of different lengths. N=6~12. Values ​​are mean ± SEM. According to two-sided Student's t-tests, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 5] The relative EXOC2 mRNA levels for EXOC2 349, measured by qRT-PCT analysis, are shown. N=3, independent derivatives. Values ​​are mean ± SEM. According to two-tailed Student's t-tests, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 6] The relative EXOC2 mRNA levels for EXOC2 3933, measured by qRT-PCT analysis, are shown. N=6. Values ​​are mean ± SEM. According to two-tailed Student's t-tests, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 7] A schematic diagram of bafilomycin A1 (Baf A1) inhibition of lysosomal V-ATPase, and therefore lysosomal function, is shown. [Figure 8]The relative EXOC2 mRNA levels measured by qRT-PCT analysis for EXOC2 3933 with inhibited and uninhibited lysosomes are shown. N=6. Values ​​are mean ± SEM. According to two-sided Student's t-test, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 9] A schematic diagram of RNase H and lysosome-based knockdown approaches for knocking down target RNA using sterically blocked ASOs is shown. [Figure 10] The relative EXOC2 mRNA levels measured by qRT-PCT analysis for sterically blocked and unblocked EXOC2 3933 are shown. N=4~6. Values ​​are mean ± SEM. According to two-tailed Student's t-tests, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 11] The relative Ku80 mRNA levels for Ku80 624 and 2802, measured by qRT-PCT analysis, are shown. N=6. Values ​​are mean ± SEM. According to two-tailed Student's t-tests, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 12] The relative TASK1 mRNA levels measured by qRT-PCT analysis for TASK1 622 and 5968 are shown. N=6. Values ​​are mean ± SEM. According to two-tailed Student's t-test, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 13] Relative SOD1 mRNA levels are shown as measured by qRT-PCT analysis in SH-SY5Y cells treated for 24 hours with 25 or 100 nM untargeted control (NTC), tofersen, or tofersen-Lamp ASO (n=3). Values ​​are mean ± SEM. By one-sided t-test, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 14]The relative PCSK9 mRNA levels are shown (n=4) as measured by qRT-PCT analysis in A549 cells treated with 50 nM inclisilane or inclisilane-Lamp for 48 hours. Values ​​are mean ± SEM. By one-sided t-test, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 15] Relative SOD1 mRNA levels are shown (n=3) as measured by qRT-PCT analysis in SH-SY5Y cells treated for 30 hours with a vehicle (mock), 30 nM tofersen-Lamp, or tofersen-Lamp derivative ASO. Values ​​are mean ± SEM. By one-sided or two-sided t-tests, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Modes for carrying out the invention]

[0097] This disclosure provides oligonucleotides, methods, and compositions for degrading target polynucleotides (e.g., RNA) using the lysosomal pathway. The disclosures herein are largely based on the discovery that antisense compounds coupled to lysosomal ligands (e.g., poly-G sequences) promote the degradation of target polynucleotides via the lysosomal pathway. While not bound by any particular theory, lysosomal ligands derived from antisense compounds interact with the lysosomal LAMP-2C protein, leading to lysosomal uptake and degradation of target polynucleotides (e.g., antisense oligonucleotides) bound to the antisense compound.

[0098] The methods described herein are not limited to the specific methods and experimental conditions disclosed herein, and it should be understood that such methods and conditions may vary. It should also be understood that the terms used herein are merely descriptive of specific embodiments and are not intended to limit them.

[0099] Furthermore, unless otherwise indicated, the experiments described herein may utilize conventional molecular and cellular biological and immunological techniques within the scope of the art. Such techniques are well known to those skilled in the art and are adequately described in the literature. For example, see Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2008), including all supplementary materials; Molecular Cloning: A Laboratory Manual (Fourth Edition) by MR. Green and J. Sambrook; and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2013). nd Please refer to the edition.

[0100] Unless otherwise defined herein, scientific and technical terms used herein have meanings generally understood by those skilled in the art. In the event of any potential ambiguity, the definitions provided herein supersede any dictionary or external definitions. Unless otherwise required by context, singular terms include plural forms, and plural terms include singular forms. The use of "or" means "and / or" unless otherwise stated. The use of the term "including," as well as other forms such as "includes" and "included," is not limited to these.

[0101] In general, the nomenclature used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is well known and commonly used in the art. The nomenclature used in relation to analytical chemistry, synthetic organic chemistry, and pharmaceuticals and medicinal chemistry described herein, as well as the experimental procedures and techniques therein, are well known and commonly used in the art.

[0102] To make this disclosure easier to understand, certain terms are defined first.

[0103] The term "nucleoside" refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine, and thymidine. Further exemplary nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and N(2),N(2)-dimethylguanosine (also known as “rare” nucleosides). The term "nucleotide" refers to a nucleoside having one or more phosphate groups linked to the sugar moiety by ester linkage. Exemplary nucleotides include nucleoside monophosphates, diphosphates, and triphosphates. The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to polymers of nucleotides linked together by phosphate diester or phosphorothioate linkages between the 5' and 3' carbon atoms.

[0104] The terms “RNA,” “RNA molecule,” or “ribonucleic acid molecule” refer to polymers of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). An RNA nucleotide refers to a single ribonucleotide. The terms “DNA,” “DNA molecule,” or “deoxyribonucleic acid molecule” refer to polymers of deoxyribonucleotides. A DNA nucleotide refers to a single deoxyribonucleotide. As used herein, the term “DNA-like” refers to conformations such as modified nucleosides or nucleotides that are similar in conformation to the corresponding unmodified DNA unit. For example, a DNA-like nucleotide may refer to a modified deoxyribonucleotide conformation that is similar to the corresponding unmodified deoxyribonucleotide. Examples of DNA-like nucleotides include, but are not limited to, 2'-deoxyribonucleotides, 2'-deoxy-2'-substituted arabinonucleotides (e.g., 2'-deoxy-2'-fluoroarabinonucleotides, also known in the art as 2'-F-ANA or FANA), and their corresponding phosphorothioate analogs. As used herein, the term "RNA-like" refers to a conformation such as a modified nucleoside or nucleotide that is similar in conformation to, for example, the conformation of the corresponding unmodified RNA unit. RNA-like structures can take the form of an A-type helix, and DNA-like structures can take the form of a B-type helix. Examples of RNA-like nucleotides include, but are not limited to, 2'-substituted RNA nucleotides (e.g., 2'-fluoro-RNA nucleotides, also known in the art as 2'F-RNA), loc nucleic acid (LNA) nucleotides (also known in the art as cross-linked nucleic acids or bicyclic nucleotides), 2'-fluoro-4'-thioarabinonucleotides (also known in the art as 4'S-FANA nucleotides), 2'-O-alkyl-RNA, and their corresponding phosphorothioate analogs.

[0105] DNA and RNA can be synthesized spontaneously (e.g., by DNA replication or DNA transcription, respectively). RNA can be modified after transcription. DNA and RNA can also be synthesized chemically. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively). "MRNA" or "messenger RNA" is a single-stranded RNA that identifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when a ribosome binds to the mRNA.

[0106] As used herein, the term “small interfering RNA” (“siRNA”) (also known in the art as “short interfering RNA”) refers to RNA (or RNA analogues) containing about 10 to 50 nucleotides (or nucleotide analogues) that can direct or mediate RNA interference. In one embodiment, an siRNA contains about 15 to 30 nucleotides or nucleotide analogues, or about 16 to 25 nucleotides (or nucleotide analogues), or about 18 to 23 nucleotides (or nucleotide analogues), or about 19 to 22 nucleotides (or nucleotide analogues) (e.g., 19, 20, 21, or 22 nucleotides or nucleotide analogues). The term “short” siRNA refers to an siRNA containing about 21 nucleotides (or nucleotide analogues), e.g., 19, 20, 21, or 22 nucleotides. The term “long” siRNA refers to an siRNA containing about 24 to 25 nucleotides, e.g., 23, 24, 25, or 26 nucleotides. Short siRNAs may, in some cases, contain fewer than 19 nucleotides, e.g., 16, 17, or 18 nucleotides, provided that the short siRNA retains its ability to mediate RNAi. Similarly, long siRNAs may, in some cases, contain more than 26 nucleotides, provided that the longer siRNA retains its ability to mediate RNAi without further processing of the short siRNA, e.g., enzymatic processing.

[0107] The terms “nucleotide analog,” “modified nucleotide,” or “modified nucleotide” refer to non-standard nucleotides, such as ribonucleotides or deoxyribonucleotides, which do not exist in nature. Exemplary modified nucleotides are modified at any position to alter the specific chemical properties of the nucleotide, while retaining the ability of the modified nucleotide to perform its intended function. Examples of nucleotide positions that can be derivatized include the 5-position, e.g., 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine; the 6-position, e.g., 6-(2-amino)propyluridine; and the 8-position of adenosine and / or guanosine, e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine. Examples of modified nucleotides include deazanucleotides, such as 7-deaza-adenosine, O and N modified (e.g., alkylated, such as N6-methyladenosine, or those known in the art) nucleotides, and other heterocyclic modified nucleotides, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug.10(4):297-310.

[0108] Modified nucleotides can also include modifications to the sugar moiety of the nucleotide. For example, the 2'OH group may be replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, COOR, or OR, where R is a substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl, aryl, etc. In another example, the ribose sugar may be replaced at the bicyclic or tricyclic moiety in, for example, locked nucleic acids, restricted ethyl, tricyclo-DNA (tcDNA), or other cross-linking or bicyclic modifications. Other possible modifications are described in U.S. Patents No. 5,858,988 and No. 6,291,438.

[0109] The phosphate group of a nucleotide can also be modified, for example, by substituting one or more oxygen atoms of the phosphate group with sulfur (e.g., a phosphorothioate), or by making other substitutions so that the nucleotide can perform its intended function, the other substitutions described in Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5):317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. 11(2):77-85, and U.S. Patent No. 5,684,143. The specific modifications referenced above (e.g., phosphate group modifications) reduce the hydrolysis rate of polynucleotides, including their analogues, in vivo or in vitro.

[0110] As used herein, the term “unmodified nucleotide” refers to a nucleotide consisting of naturally occurring nucleic acid bases, a sugar moiety, and internucleoside links. In some embodiments, the unmodified nucleotide is an RNA nucleotide (i.e., a β-D-ribonucleoside) or a DNA nucleotide (i.e., a β-D-deoxyribonucleoside).

[0111] The term "oligonucleotide" refers to a short polymer of nucleotides and / or modified nucleotides. As mentioned above, oligonucleotides can be linked by non-phosphodiester linkages, which results in a lower hydrolysis rate compared to oligonucleotides linked by phosphodiester linkages. For example, the nucleotides of an oligonucleotide can include triazole, amide, carbamate, methylenediol, ethylenediol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoramidate, phosphonate, and / or phosphorothioate linkages. Modification or alteration of an oligonucleotide can further include the addition of a non-nucleotide substance to the end(s) or interior (with one or more nucleotides of the oligonucleotide). Oligonucleotides can contain lysosomal ligands.

[0112] As used herein, the term “antisense oligonucleotide” refers to an oligonucleotide molecule capable of binding to intracellular RNA by Watson-Crick base pairing. Depending on the sequence and chemistry of the antisense oligonucleotide, this interaction can result in either silencing of a target gene (i.e., a decrease in the expression level of mature mRNA and / or protein from that gene) or activation of a target gene (i.e., an increase in the expression level of mature mRNA and / or protein from that gene). The antisense oligonucleotides of this disclosure focus on the activation of gene expression that can be achieved by utilizing different mechanisms. Some antisense oligonucleotides are designed to recruit RNase H to cleave their target RNA. RNase H is a family of sequence-specific endonuclease enzymes that catalyze the cleavage of RNA in RNA / DNA substrates via a hydrolysis mechanism. In certain embodiments, the antisense oligonucleotides of this disclosure trigger RNase H-mediated cleavage of pre-mRNA targets (e.g., EXOC2, Ku80, and Task1 pre-mRNA), which can be coupled with the activation of overall target gene expression (e.g., EXOC2, Ku80, and Task1 gene expression). Other antisense oligonucleotides, known as steric barriers, are designed not to trigger the cleavage of their targets, but to block interactions with cellular factors. For example, these cellular factors can regulate splicing, block interactions with non-coding RNA or RNA-binding proteins, stabilize mRNA to extend its half-life, or improve the translation efficiency of mRNA.

[0113] Antisense oligonucleotides designed to recruit RNase H are often designed as “gapmers.” The term “gapmer” refers to a chimeric antisense oligonucleotide in which an internal region containing multiple nucleosides that assist in RNase H cleavage is positioned between one or more external regions containing one or more nucleosides, where these nucleosides, including the internal region, are chemically distinct from the nucleosides or multiple nucleosides, including the external region. The internal region may be called a “gap segment,” and the external region may be called a “wing segment.” A “chimeric antisense oligonucleotide” means an antisense oligonucleotide having at least two chemically distinct regions.

[0114] As used herein, the term “lysosomal ligand” refers to a ligand that interacts with lysosomes. While not bound by any particular theory, lysosomal ligands interact with the lysosomal LAMP-2C protein, triggering lysosomal uptake and degradation of target RNA bound to oligonucleotides containing the lysosomal ligand. In some embodiments, the lysosomal ligand is a poly-G ligand or sequence.

[0115] As used herein, "poly-G ligand" or "poly-G sequence" is a polynucleotide sequence containing two or more G nucleotides and lacking complementarity to the target polynucleotide of the oligonucleotide to which the poly-G sequence binds.

[0116] In some embodiments, the poly-G sequence contains 2 to 30 G nucleotides. In some embodiments, the poly-G sequence contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 5 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 6 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 7 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 8 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 9 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 10 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 11 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 12 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 13 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 14 G nucleotides. In some embodiments, the poly-G sequence contains or consists of 15 G nucleotides.

[0117] In some embodiments, the poly-G sequence is ligated to the 3' end of the oligonucleotide of the disclosure. In other embodiments, the poly-G sequence is ligated to the 5' end of the oligonucleotide of the disclosure. In other embodiments, the poly-G sequence is ligated to both the 5' and 3' ends of the oligonucleotide of the disclosure.

[0118] In some embodiments, the poly-G sequence is single-stranded.

[0119] In some embodiments, the poly-G sequence is bound to a protein on the surface of a lysosome. In some embodiments, the poly-G sequence is bound to a lysosome-associated membrane glycoprotein (LAMP). In some embodiments, the LAMP is LAMP2C.

[0120] In some embodiments, the poly-G sequence contains consecutive G nucleotides. In some embodiments, the poly-G sequence contains or consists of 5 to 15 consecutive G nucleotides. In some embodiments, the poly-G sequence contains one or more non-G nucleotides (e.g., A, T, U, or C) within the poly-G sequence. In some embodiments, the oligonucleotide contains two or more poly-G sequences having one or more non-G nucleotides (e.g., A, T, U, or C) between them. For example, the oligonucleotide may, but is not limited to, have a first poly-G sequence of 5 to 10 consecutive G nucleotides, one or more non-G nucleotides (e.g., A, T, U, or C), and a second poly-G sequence of 5 to 10 consecutive G nucleotides at its 3' end (5' to 3').

[0121] In some embodiments, the poly-G sequence includes non-contiguous G nucleotides.

[0122] In some embodiments, the polyG sequence includes or consists of (dG)(dG)(dG)(dG)(dG). In some embodiments, the polyG sequence includes or consists of (dG)(dG)(dG)(dG)(dG)(dG). In some embodiments, the polyG sequence includes or consists of (dG)(dG)(dG)(dG)(dG)(dG)(dG). In some embodiments, the polyG sequence includes or consists of (dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG). In some embodiments, the polyG sequence includes or consists of (dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG). In some embodiments, the polyG sequence includes or consists of (dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG). In some embodiments, the polyG sequence includes or consists of (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dN)(dG), where each dN corresponds individually to a deoxyribonucleotide of dA, dT, or dC. In some embodiments, the polyG sequence includes or consists of (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dN)(dG)(dG), where each dN corresponds individually to a deoxyribonucleotide of dA, dT, or dC. In some embodiments, the polyG sequence comprises or consists of (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dN)(dG)(dG), where each dN corresponds individually to a deoxyribonucleotide of dA, dT, or dC.

[0123] As used herein, the term “functional moiety” refers to the moiety linked to an oligonucleotide. The functional moiety is linked to the 5' or 3' end of the oligonucleotide. The functional moiety may include an N-acetylgalactosamine (GalNAc) moiety and / or a hydrophobic moiety. The hydrophobic moiety may be a fatty acid, a steroid, a secosteroid, a lipid, a ganglioside, a nucleoside analog, an endocannabinoid, a vitamin, or a mixture thereof. Steroids may be cholesterol and lithocholic acid (LCA). Fatty acids may be eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanic acid (DCA).

[0124] As used herein, the term “target polynucleotide” refers to a polynucleotide that is sufficiently complementary to the oligonucleotides of this disclosure in order to mediate lysosome targeting via a poly-G sequence. Exemplary target polynucleotides include mRNA and viral RNA.

[0125] As used herein, the term “target gene” refers to a gene whose expression is substantially silenced, restored, or increased. In certain embodiments, target gene expression is silenced, restored, or increased to wild-type levels via direct base-pairing interactions with the target sequence and by oligonucleotides that silence expression via lysosomal pathways (e.g., EXOC2, Ku80, and Task1 mRNA target sequences). In certain embodiments, target gene expression is silenced, restored, or increased to wild-type levels via RNA silencing, for example, by cleaving the transcript corresponding to the target gene or by translational repression of the target gene. While not theoretically bound, cleaving the target transcript can increase the level of productive transcription of the target gene. For example, expressing an allele of the target gene can form a deficient pre-mRNA (e.g., containing a disease-contributing nucleotide repeat region). Target gene expression can be restored by cleaving and degrading the deficient pre-mRNA derived from the deficient allele, thereby releasing the transcription mechanism and triggering transcription of the non-deficient target gene allele. The term “non-target gene” refers to a gene whose expression is not substantially increased, restored, or silenced. For example, the target genes in this disclosure are EXOC2, Ku80, and Task1, and the non-target genes in this disclosure are genes other than EXOC2, Ku80, and Task1. In one embodiment, the polynucleotide sequences of the target gene and the non-target gene (e.g., the mRNA encoded by the target gene and the non-target gene) may differ by one or more nucleotides. In another embodiment, the target and non-target genes may differ by one or more polymorphisms (e.g., single nucleotide polymorphisms or SNPs). In another embodiment, the target and non-target genes may share less than 100% sequence identity. In another embodiment, the target and non-target genes may share less than 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 86%, 85%, 80%, 75%, or 70% sequence identity.In another embodiment, the non-target gene may be a homolog (e.g., an ortholog or paralog) of the target gene.

[0126] The term “antisense activity” means any detectable or measurable activity resulting from the hybridization of an antisense compound to its target nucleic acid. In some embodiments, antisense activity is an increase in the amount or expression of the target nucleic acid or the protein encoded by such target nucleic acid. “Antisense compound” means an oligomeric compound that can undergo hybridization to a target nucleic acid by hydrogen bonding. As used herein, “antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleic acid base sequence that enables hybridization to a corresponding region or segment of the target nucleic acid.

[0127] The term "antisense inhibition" refers to a reduction in the level of the target nucleic acid in the presence of an antisense oligonucleotide having a sequence sufficiently complementary to the target nucleic acid, compared to the level of the target nucleic acid in the absence of an antisense compound. The target nucleic acid can be any nucleic acid. For example, the target nucleic acid in this disclosure may be EXOC2, Ku80, Task1, SOD1, or PCSK9 transcript. In certain embodiments, the target nucleic acid is EXOC2, Ku80, Task1, SOD1, or PCSK9 pre-mRNA.

[0128] The term "target recognition sequence" refers to the portion of an antisense compound that recognizes a target nucleic acid. The target recognition sequence has a nucleic acid base sequence that enables hybridization to a corresponding region or segment of the target nucleic acid.

[0129] The term "conserved region" refers to a portion or more of a conserved nucleic acid sequence, i.e., a portion or more of a nucleic acid sequence that has similar or identical sequences across species. Conserved regions can be computationally identified, for example, using any sequence alignment software available in the art.

[0130] As used herein, the term “sufficiently complementary” means that an antisense compound has a sequence (e.g., an antisense oligonucleotide having a target recognition sequence) sufficient to bind to a desired target transcript (e.g., an EXOC2, Ku80, or Task1 transcript) and to increase, restore, or silence the expression of the EXOC2, Ku80, or Task1 gene. For example, a target recognition sequence having at least 90% complementarity to a target nucleic acid sequence (e.g., a portion of an EXOC2, Ku80, or Task1 transcript) can be sufficiently complementary to increase, restore, or silence the expression of the EXOC2, Ku80, or Task1 gene. The term “fully complementary” refers to, for example, a target recognition sequence having 100% complementarity to a target nucleic acid sequence. Complementary nucleic acid molecules hybridize with each other. The term “hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include an antisense compound and a target nucleic acid.

[0131] As used herein, “complementary region” refers to a portion of an antisense oligonucleotide that is complementary to the target transcript (e.g., EXOC2, Ku80, or Task1 transcript). For example, an antisense oligonucleotide with a length of 18 nucleotides may include a portion of 12 consecutive nucleotides that is complementary to the target transcript. In certain embodiments, the antisense oligonucleotide is complementary to the target transcript throughout its entire length.

[0132] As used herein, “administer” or “administration” means the act of injecting or otherwise physically delivering a substance (e.g., an antisense compound provided herein) to a patient when the substance is outside the body. The antisense oligonucleotides described herein can be delivered to the central nervous system of a patient. The central nervous system includes the brain and spinal cord. Methods of administration to the central nervous system include, but are not limited to, intrathecal, intraventricular, or intrastriatal injection or delivery, and / or any other physical delivery methods described herein or known in the art. Intraventricular injection may include administration using an Omaya reservoir. In some embodiments, the antisense oligonucleotides described herein can be administered systemically to a patient (e.g., intravenously, subcutaneously, or intramuscularly). These compounds may be designed to enter the central nervous system or to be activated in muscle (including skeletal muscle or cardiac muscle) or other tissues such as the pancreas.

[0133] When a disease or its symptoms are under control or treatment, administration of the substance is typically performed after the onset of the disease or its symptoms. When a disease or its symptoms are being prevented, administration of the substance is typically performed before the onset of the disease or its symptoms and may be continued for an extended period to delay the appearance or reduce the severity of disease-related symptoms, such as damage to the tissues and airways involved.

[0134] As used herein, the term “composition” is intended to include not only products that optionally contain a given component (e.g., an antisense compound provided herein) in a predetermined amount, but also any products that result directly or indirectly from the combination of a given component in a predetermined amount, either optionally.

[0135] "Effective amount" means the amount of active pharmaceutical agent (e.g., the antisense compound of this disclosure) that is sufficient to produce the desired physiological outcome in an individual requiring the drug. The effective amount may vary between individuals depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated, the formulation of the composition, the assessment of the individual's medical condition, and other relevant factors.

[0136] As used herein, the terms “subject” and “patient” are used synonymously. As used herein, the subject may be an animal other than a primate (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) or a mammal such as a primate (e.g., monkeys and humans). In certain embodiments, the term “subject” refers to a vertebrate such as a mammal. Mammals include, but are not limited to, humans, non-human primates, wild animals, feral animals, livestock, sports animals, and pets. In one embodiment, the subject is a mammal such as a human having EXOC2-related disorder, Ku80-related disorder, and / or Task1-related disorder. In another embodiment, the subject is a mammal such as a human at risk of developing EXOC2-related disorder, Ku80-related disorder, and / or Task1-related disorder.

[0137] As used herein, the term “therapy” refers to any protocol, method and / or agent that may be used in the prevention, management, treatment and / or improvement of a disease or its associated symptoms, such as EXOC2-related disorders, Ku80-related disorders, and / or Task1-related disorders. In some embodiments, the term “therapy” refers to any protocol, method and / or agent that may be used in the modulation of the immune response to an infection or its associated symptoms in a subject. In some embodiments, the term “therapy” (singular and plural) refers to biological therapies, supportive therapies and / or other therapies known to those skilled in the art, such as healthcare professionals, that are useful in the prevention, management, treatment and / or improvement of a disease or its associated symptoms, such as EXOC2-related disorders, Ku80-related disorders, and / or Task1-related disorders. In other embodiments, the term “therapy” (singular and plural) refers to biological therapies, supportive therapies and / or other therapies known to those skilled in the art, that are useful in the modulation of the immune response to an infection or its associated symptoms in a subject.

[0138] As used herein, the terms “to treat,” “treatment,” and “to treat” mean a reduction or improvement in the progression, severity, and / or duration of a disease or its associated symptoms, such as EXOC2-related disorders, Ku80-related disorders, and / or Task1-related disorders, resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents, such as antisense oligonucleotides provided herein). As used herein, the term “to treat” may also mean altering the disease course of the subject being treated. The therapeutic effect of treatment includes, but is not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improving or reducing the disease state, and achieving remission or an improved prognosis.

[0139] As used herein, the term "EXOC2" refers to the gene encoding component 2 of the protein exocyst complex. Protein EXOC2 is a component of the exocyst complex, a multiprotein complex essential for the polarization targeting of extracellular vesicles to specific docking sites on the cell membrane. The human EXOC2 gene corresponds to NG_047166.1 in the NCBI RefSeq database.

[0140] As used herein, the term "Ku80" refers to the protein encoded by the XRCC5 gene. Ku80 is a component of the Ku heterodimer with Ku70, and it binds to the ends of DNA double-strand breaks and is required for the non-homologous end-joining (NHEJ) pathway of DNA repair. The human XRCC5 gene corresponds to NG_029780.1 in the NCBI RefSeq database. The term Ku80 is also used herein to refer to the gene and mRNA encoding Ku80. Thus, oligonucleotides targeting Ku80 and Ku80 mRNA (including pre-mRNA) are used.

[0141] As used herein, the terms "Task1" or "KCNK3" refer to the gene encoding the potassium channel subfamily K member 3 protein. Task1 is one of the members of the superfamily of potassium channel proteins containing two pore-forming P domains. The human KCNK3 gene corresponds to NG_033884.1 in the NCBI RefSeq database. SOD1 inhibitors, such as inclisilane, are used to treat hyperlipidemia or reduce low-density lipoprotein cholesterol (LDL-C) in subjects.

[0142] As used herein, the term "SOD1" refers to the gene encoding the superoxide dismutase 1 protein. SOD1 is a mammalian enzyme that catalyzes the removal of superoxide radicals. The human SOD1 gene corresponds to NG_008689.1 in the NCBI RefSeq database. SOD1 inhibitors, such as tofersen, are used to treat amyotrophic lateral sclerosis (ALS) in subjects.

[0143] As used herein, the term "PCSK9" refers to the gene encoding the proprotein convertase subtilisin / kexin 9 protein. PCSK9 binds to and degrades low-density lipoprotein particles (LDL) receptors. The human PCSK9 gene corresponds to NG_009061.1 in the NCBI RefSeq database. PCSK9 inhibitors, such as inclisilane, are used to treat hyperlipidemia or reduce low-density lipoprotein cholesterol (LDL-C) in subjects.

[0144] Antisense compounds This disclosure provides antisense compounds linked to poly-G sequences disclosed herein for targeting polynucleotides to cellular lysosomes for targeted degradation.

[0145] The disclosure also provides antisense compounds that can reduce the expression of EXOC2, Ku80, and Task1 genes by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, or more.

[0146] In certain embodiments, an antisense compound capable of reducing the expression of a target polynucleotide (e.g., EXOC2, Ku80, and Task1 gene expression) has chemically modified subunits arranged in a pattern or motif to confer antisense compound properties such as enhanced activity, increased binding affinity to the target nucleic acid, or resistance to degradation by in vivonucleases.

[0147] In some embodiments, the antisense compounds of the present disclosure are antisense oligonucleotides. Chimeric antisense oligonucleotides typically contain at least one region modified to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity to a target nucleic acid, and / or increased activity. The second region of the chimeric antisense compound can optionally serve as a substrate for RNase H, a cellular endonuclease that cleaves the RNA strand of an RNA:DNA double helix. In some embodiments, the antisense compounds of the present disclosure are chimeric antisense oligonucleotides having a gapmer motif. In the gapmer, an inner region having multiple nucleotides supporting RNase H cleavage is positioned between an outer region having multiple nucleotides chemically distinct from the nucleoside of the inner region.

[0148] In some embodiments, the disclosure provides antisense oligonucleotides having a target recognition sequence that is sufficiently complementary to EXOC2, Ku80, and / or the Task1 transcript or a portion thereof, in order to induce cleavage of the EXOC2, Ku80, and Task1 transcript by RNase H. The target recognition sequence of the antisense oligonucleotide may be the entire length of the antisense oligonucleotide or a portion thereof. In some embodiments, the antisense oligonucleotide includes a gapmer motif.

[0149] In the case of antisense compounds having a gapmer motif, the gap segment generally serves as a substrate for endonuclease cleavage, while the wing segment contains a modified nucleoside. In certain embodiments, the gapmer region is distinguished by the type of sugar moiety that constitutes the individual distinct regions. Types of sugar moieties used to distinguish the gapmer region include, in some embodiments, β-D-ribonucleosides, β-D-deoxyribonucleosides, 2'-modified nucleosides (such as 2'-MOE and 2'-O-CH3 (i.e., OMe)), and bicyclic sugar-modified nucleosides (such as 4'-(CH2) n Examples include those having an O-2' crosslink (wherein n=1 or n=2). In some embodiments, the wing segments of the gapmer contain one or more tricyclo-DNA (tcDNA) modifications. In some embodiments, each distinct region contains a uniform sugar moiety. In some embodiments, each wing segment contains a mixture of different nucleotide modifications. For example, in one embodiment, LNA modification and 2'-MOE modification can be used in combination with one antisense compound. In one embodiment, LNA modification and 2'-OM ethyl modification can be used in combination with one antisense compound. In one embodiment, LNA modification and 2'-deoxy modification can be used in combination with one antisense compound. In one embodiment, LNA modification and tricyclo-DNA modification can be used in combination with one antisense compound. In one embodiment, 2'-MOE modification and tricyclo-DNA modification can be used in combination with one antisense compound.

[0150] The gapmer motif can be described using the formula "ABC", where "A" represents the length of the 5' wing region, "B" represents the length of the gap region, and "C" represents the length of the 3' wing region. Thus, in some embodiments, the antisense oligonucleotide of the present disclosure is expressed by formula It has ABC.

[0151] In this specification, the gapmer referred to as "ABC" has a configuration such that the gap segment is directly adjacent to both the 5' wing segment and the 3' wing segment. Therefore, there are no intervening nucleotides between the 5' wing segment and the gap segment, nor between the gap segment and the 3' wing.

[0152] In some embodiments, the 5' wing region represented by "A" contains about 0 to about 8 modified nucleotides, for example, about 1 to about 6 modified nucleotides. For example, the 5' wing region represented by "A" can have a length of 0, 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides. In some embodiments, the 3' wing region represented by "C" contains about 0 to about 8 modified nucleotides, for example, about 1 to about 6 modified nucleotides. For example, the 3' wing region represented by "C" can have a length of 0, 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides. In some embodiments, "A" and "C" are identical, and in some embodiments, they are different.

[0153] In some embodiments, the gap region represented by "B" contains about 5 to about 18 DNA nucleotides and / or DNA-like nucleotides, for example, about 5 to about 12 DNA nucleotides and / or DNA-like nucleotides. For example, the gap region represented by "B" can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 DNA nucleotides and / or DNA-like nucleotides of length. Thus, antisense oligonucleotides of this disclosure having a target recognition sequence having formula "ABC" include, for example, 1-10-1 (i.e., 1 nucleotide-10 nucleotides-1 nucleotide), 1-10-1, 1-11-1, 1-12-1, 2-8-2, 2-9-2, 2-10-2, 2-11-2, 2-12-2, 3-6-3, 3-7-3, 3-8-3, 3-9-3, 3-10-3, Examples of gapmer forms include, but are not limited to, 3-11-3, 3-12-3, 4-6-4, 4-7-4, 4-8-4, 4-9-4, 4-10-4, 4-11-4, 4-12-4, 5-6-5, 5-7-5, 5-8-5, 5-9-5, 5-10-5, 5-11-5, 5-12-5, 6-6-6, 6-7-6, 6-8-6, 6-9-6, 6-10-6, 6-11-6, or 6-12-6. The wings can also be of different lengths, such as 1-10-6, 3-9-5, 7-9-2, 4-10-5, or other asymmetrical combinations of wing lengths adjacent to the central DNA gap. In a particular embodiment, the gapmer of “ABC” is at least 12 nucleotides long. In a particular embodiment, “B” is at least 6 nucleotides long. Those skilled in the art can identify additional asymmetrical combinations of wing lengths.

[0154] In certain embodiments, the antisense compound targeting EXOC2, Ku80, or Task1 nucleic acid has a 5-9-4 gapmer form. In some embodiments, the antisense compound is an antisense oligonucleotide having a 5-9-4 form target recognition sequence that is sufficiently complementary to the EXOC2, Ku80, or Task1 transcript, or a portion thereof, in order to induce cleavage of the EXOC2, Ku80, or Task1 transcript by RNase H. In some embodiments, the target recognition sequence has the formula "ABC", where "A" comprises about 2 to 6 modified nucleotides, "B" comprises about 6 to 12 DNA nucleotides and / or DNA-like nucleotides, and "C" comprises about 2 to 6 modified nucleotides. In some embodiments, the target recognition sequence has the formula "ABC", where "A" comprises 5 modified nucleotides, "B" comprises 9 DNA nucleotides and / or DNA-like nucleotides, and "C" comprises 4 modified nucleotides. In some embodiments, the target recognition sequence has the formula "ABC", where "A" comprises 2 to 6 2'-O-(2-methoxyethyl)(MOE) modified nucleotides, "B" comprises 6 to 12 DNA nucleotides and / or DNA-like nucleotides, and "C" comprises 2 to 6 2'-O-(2-methoxyethyl)(MOE) modified nucleotides. In some embodiments, the target recognition sequence has the formula "ABC", where "A" comprises 5 2'-O-(2-methoxyethyl)(MOE) modified nucleotides, "B" comprises 9 DNA nucleotides and / or DNA-like nucleotides, and "C" comprises 4 2'-O-(2-methoxyethyl)(MOE) modified nucleotides.

[0155] In some embodiments, antisense compounds targeting EXOC2, Ku80, or Task1 nucleic acids have a "wingmer" motif. The wingmer motif can be described using the formula "XY" or "YX," where "X" represents the length of the wing region and "Y" represents the length of the gap region. Thus, in some embodiments, the antisense oligonucleotides of the present disclosure have the formula XY, or YX is the number.

[0156] As used herein, a wingmer described as "XY" or "YX" is configured such that the gap segment is positioned directly adjacent to the wing segment. Therefore, there are no intervening nucleotides between the wing segment and the gap segment. Non-limiting examples of wingmer configurations of the antisense compounds of this disclosure include, for example, 1-15, 1-17, 1-19, 2-15, 2-17, 2-19, 2-22, 3-13, 3-17, 3-20, 3-21, 3-22, 4-12, 4-14, 4-16, 4-18, 4-19, 4-21, 5-11, 5-13, 5-14, 5-15, 5-16, 5-18, or 5-20.

[0157] In some embodiments, antisense compounds targeting EXOC2, KU80, or TASK1 nucleic acids have a gap-expanded motif. As used herein, “gap-expanded” refers to an antisense compound having a gap segment of 12 or more consecutive DNA nucleotides and / or DNA-like nucleotides adjacent to a wing region. In the case of a gap-expanded gapmer, the gapmer includes a gap region having 12 or more consecutive DNA nucleotides and / or DNA-like nucleotides located between the 5' wing segment and the 3' wing segment, and adjacent to them. In the case of a gap-expanded wingmer, the wingmer includes a gap region having 12 or more consecutive DNA nucleotides and / or DNA-like nucleotides located directly adjacent to the wing segment.

[0158] A nucleoside is a base-sugar combination. The nucleic acid base (also known as the base) portion of a nucleoside is usually a heterocyclic base portion. A nucleotide is a nucleoside that further contains a phosphate group covalently linked to the sugar portion of the nucleoside. In the case of nucleosides containing pentofuranosyl sugars, the phosphate group can be linked to the 2', 3', or 5' hydroxyl portion of the sugar. Linear polymeric oligonucleotides are formed by covalently linking adjacent nucleosides to each other. Generally, within the oligonucleotide structure, the phosphate group is considered to form the internucleoside linkage of the oligonucleotide.

[0159] Modifications to antisense compounds include substitutions or alterations of nucleoside junctions, sugar moieties, or nucleic acid bases. Modified antisense compounds are often preferred over native compounds due to desirable properties such as enhanced cellular uptake, improved affinity for nucleic acid targets, increased stability in the presence of nucleases, and increased inhibitory activity.

[0160] Chemically modified nucleosides can also be used to increase the binding affinity of truncated or cleaved antisense oligonucleotides to their target nucleic acids. As a result, comparable results can often be obtained with short antisense compounds that have such chemically modified nucleosides.

[0161] The naturally occurring nucleoside linkages in RNA and DNA are phosphodiester links from 3' to 5'. Antisense compounds with one or more modified (i.e., non-naturally occurring) nucleoside linkages are often preferred over antisense compounds with naturally occurring nucleoside linkages due to desirable properties such as enhanced cellular uptake, improved affinity for nucleic acid targets, and increased stability in the presence of nucleases.

[0162] Oligonucleotides having modified nucleoside linkages include nucleoside linkages that retain a phosphorus atom and nucleoside linkages that do not contain a phosphorus atom. Typical phosphorus-containing nucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known.

[0163] In certain embodiments, an antisense compound targeting EXOC2, KU80, or TASK1 nucleic acid comprises one or more modified nucleoside links. In certain embodiments, the modified nucleoside links are phosphorothioate links. In certain embodiments, each nucleoside link in the antisense compound is a phosphorothioate nucleoside link.

[0164] The antisense compounds of this disclosure may optionally contain one or more nucleosides that are modified with sugar groups. Such sugar-modified nucleosides can confer enhanced nuclease stability, increased binding affinity, or any other beneficial biological properties to the antisense compounds. In certain embodiments, the nucleoside comprises a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include the addition of substituents (5' substituent, 2' substituent, crosslinking of ring atoms to form bicyclic nucleic acids (BNAs), S, N(R), or C(R) 1 )(R 2 )(R=H, C1-C 12Examples of chemically modified sugars include, but are not limited to, substitution of the ribosyl ring oxygen atom with alkyl or protecting groups, and combinations thereof. Examples include 2'-F-5'-methyl-substituted nucleosides (see PCT international application WO2008 / 101157, published August 21, 2008, for other disclosed 5',2'-bis-substituted nucleosides), or substitution of the ribosyl ring oxygen atom with S and further substitution at the 2' position (see US Patent Application US2005-0130923, published June 16, 2005), or alternatively, 5'-substitution of BNA (see PCT international application WO2007 / 134181, disclosed November 22, 2007, where LNA is substituted with, for example, a 5'-methyl group or a 5'-vinyl group).

[0165] Examples of nucleosides having a modified sugar moiety include, but are not limited to, nucleosides containing substituents such as 5'-vinyl, 5'-methyl(R or S), 4'-S, 2'-F (i.e., 2'-fluoro), 2'-OCH3 (i.e., 2'-O-methyl), and 2'-O(CH2)2OCH3 (i.e., 2'-O-methoxyethyl). Substituents at the 2' position also include allyl, amino, azide, thio, O-allyl, O-C1-C10 alkyl, OCF3, O(CH2)2SCH3, and O(CH2)2-ON(R m )(R n ), and O-CH2-C(=O)-N(R m )(R n ) can be selected from, and in the formula, each R m and R n This is independently H, or a substituted or unsubstituted C1-C10 alkyl group. 2'-modified nucleotides, such as 2'-O-methylRNA, 2'-O-methoxyethylRNA, 2'-fluoroRNA, and others as envisioned by those skilled in the art, are useful in this disclosure.

[0166] Examples of bicyclic nucleosides (BNAs) include, but are not limited to, nucleosides containing a bridge between the 4'-ribosyl ring atom and the 2'-ribosyl ring atom. BNAs containing a bridge between the 4'-ribosyl ring atom and the 2'-ribosyl ring atom may be called locked nucleic acids (LNAs) and are often referred to as inaccessible RNAs. As used herein, the terms “locked nucleotide” or “locked nucleic acid (LNA)” include nucleotides in which the 2'-deoxyribose sugar moiety is modified by the introduction of a structure containing a heteroatom bridge from the 2' carbon atom to the 4' carbon atom. The term “unlocked nucleotide” includes nucleotides that do not contain a bridge structure in the ribose sugar moiety. Therefore, this term includes DNA and RNA nucleotide monomers (phosphorylated adenosine, guanosine, uridine, cytidine, deoxyadenosine, deoxyguanosine, deoxythymidine, deoxycytidine) and their derivatives, as well as other nucleotides having a 2'-deoxy-erythro-pentofuranosyl sugar moiety or a ribo-pentofuranosyl moiety. In certain embodiments, the antisense compounds provided herein comprise one or more BNA nucleosides, the crosslinks being 4'-(CH2)-O-2'(LNA), 4'-(CH2)-S-2', 4'-(CH2)-O-2'(LNA), 4'-(CH2)2-O-2'(ENA), 4'-C(CH3)2-O-2' (see PCT / US2008 / 068922), 4'-CH(CH3)-O-2' and 4'-CH(CH2OCH3)-O-2' (see U.S. Patent No. 7,399,845 issued July 15, 2008), 4'-CH2- The formula includes one of the following: N(OCH3)-2' (see PCT / US2008 / 064591), 4'-CH2-ON(CH3)-2' (see U.S. Patent Application Publication US2004-0171570, published September 2, 2004), 4'-CH2-N(R)-O-2' (see U.S. Patent No. 7,427,672, issued September 23, 2008), 4'-CH2-C(CH3)-2', and 4'-CH2-C(=CH2)-2' (see PCT / US2008 / 066154), where R is independently H, C1-C12 alkyl, or a protecting group.Each of the aforementioned BNAs includes various stereochemical sugar configurations, such as α-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT / DK98 / 00393, published as WO99 / 14226 on March 25, 1999).

[0167] In some embodiments, the antisense compounds provided herein comprise one or more 2',4'-restricted nucleotides. For example, the antisense compounds provided herein comprise one or more restricted ethyl (cEt) or restricted methoxyethyl (cMOE) nucleotides. In some embodiments, the antisense compounds provided herein are antisense oligonucleotides comprising one or more restricted ethyl (cEt) nucleotides. The terms “restricted ethyl” and “ethyl-restricted” are used interchangeably.

[0168] In certain embodiments, the nucleoside is modified by substitution of the ribosyl ring with a sugar substitute. This modification includes, but is not limited to, substitution of the ribosyl ring with alternative ring systems (often referred to as DNA analogs), such as a morpholino ring, cyclohexenyl ring, cyclohexyl ring, or tetrahydropyranyl ring, which have one of the following formulas: [ka]

[0169] In certain embodiments, the antisense oligonucleotide may comprise a morpholino ring linked by phosphorodiamidate linkages. These may be referred to as PMO oligomers or phosphorodiamidate morpholino oligomers. In certain such embodiments, the backbone of these oligonucleotides may be uncharged. In other embodiments, one or more of the phosphorodiamidate linkages may comprise a charged moiety.

[0170] Many other bicyclo and tricyclosaccharide substitute ring systems that can be used to modify nucleosides for incorporation into antisense compounds are also known in the art (see, for example, Leumann, JC, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854; Ito, KR, Obika, S., Recent Advances in Medicinal Chemistry of Antisense Oligonucleotides. In Comprehensive Medicinal Chemistry, 3rd edition, Elsevier: 2017). Such ring systems may undergo various additional substitutions to enhance their activity.

[0171] Methods for preparing modified sugars are well known to those skilled in the art. In nucleotides having a modified sugar moiety, the nucleic acid base moiety (natural, modified, or a combination thereof) is maintained for hybridization with a suitable nucleic acid target.

[0172] In certain embodiments, an antisense compound targeting EXOC2, Ku80, or Task1 nucleic acid comprises one or more types of modified nucleotides. In one embodiment, an antisense compound targeting EXOC2, Ku80, or Task1 nucleic acid comprises a 2'-modified nucleotide. In one embodiment, an antisense compound targeting EXOC2, Ku80, or Task1 nucleic acid comprises 2'-O-methylRNA, 2'-O-methoxyethylRNA, or 2'-fluoroRNA. In one embodiment, an antisense compound targeting EXOC2, Ku80, or Task1 nucleic acid comprises tricyclo-DNA (tcDNA). Tricyclo-DNA belongs to a class of constrained DNA analogs that exhibit improved hybridization ability to complementary RNA. See, for example, Ittig et al., Nucleic Acids Res. 32:346-353 (2004), Ittig et al., Prague, Academy of Sciences of the Czech Republic. 7:21-26 (Coll.Symp.Series, Hocec, M., 2005), Ivanova et al., Oligonucleotides 17:54-65 (2007), Renneberg et al., Nucleic Acids Res. 30:2751-2757 (2002), Renneberg et al., Chembiochem. 5:1114-1118 (2004), and Renneberg et al., JACS. 124:5993-6002 (2002). In one embodiment, an antisense compound targeting EXOC2, Ku80, or Task1 nucleic acid comprises a lock nucleotide, an ethyl-restricted nucleotide, or an alpha-L lock nucleic acid. Various alpha-L lock nucleic acids are well known to those skilled in the art and are described, for example, in Sorensen et al., J.Am.Chem.Soc.(2002)124(10):2164-2176.

[0173] In certain embodiments, the antisense compound targeting EXOC2, Ku80, or Task1 nucleic acid is fully chemically modified, i.e., all nucleotides are chemically modified. In certain embodiments, each nucleotide includes a 2'-O-(2-methoxyethyl)(MOE) modification. In certain embodiments, each nucleotide includes a tricyclo-DNA modification. In certain embodiments, the antisense compound targeting EXOC2, Ku80, or Task1 nucleic acid includes a mixture of tricyclo-DNA modifications and 2'-O-(2-methoxyethyl)(MOE) modifications, and all nucleotides in the antisense compound are either tcDNA or MOE.

[0174] In certain embodiments, an antisense compound targeting EXOC2, Ku80, or Task1 nucleic acid comprises one or more modified nucleotides having a modified sugar moiety. In some embodiments, the modified nucleotides are lock nucleotides. In certain embodiments, the lock nucleotides are arranged in a gapmer motif, e.g., a 3-9-3 gapmer format, with nine non-lock nucleotides flanking three lock nucleotides on each side.

[0175] Modifications or substitutions of nucleic acid bases (or bases) are structurally distinguishable from naturally occurring or synthetically unmodified nucleic acid bases, but are also functionally interchangeable. Both natural and modified nucleic acid bases can participate in hydrogen bonding. Such nucleic acid base modifications can confer nuclease stability, binding affinity, or other beneficial biological properties to antisense compounds. Modified nucleic acid bases include, for example, synthetic and natural nucleic acid bases such as 5-methylcytosine (5-me-C). Certain nucleic acid base substitutions, including 5-methylcytosine substitutions, are useful for increasing the binding affinity of antisense compounds to target nucleic acids. For example, 5-methylcytosine substitutions have been shown to increase the stability of nucleic acid double helix by 0.6–1.2°C (Sanghvi, YS, Crooke, ST. Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276–278).

[0176] Additional modified nucleic acid bases 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, 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 ( Examples include pseudouracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, such as 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

[0177] The heterocyclic base moieties may also include those in which the purine or pyrimidine base is substituted with other heterocyclic bases, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Nucleic acid bases useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.

[0178] In certain embodiments, an antisense compound targeting EXOC2, Ku80, or Task1 nucleic acid comprises one or more modified nucleotides having a modified sugar moiety. In some embodiments, the modified nucleotide is a lock nucleotide. In certain embodiments, the lock nucleotide is arranged in a gapmer motif, e.g., a 3-9-3 gapmer format, with nine non-lock nucleotides flanking three lock nucleotides on each side. In certain embodiments, an antisense compound targeting EXOC2, Ku80, or Task1 nucleic acid comprises one or more modified nucleotides. In some embodiments, the modified nucleotide is 5-methylcytosine. In certain embodiments, each cytosine is 5-methylcytosine. In some embodiments, the modified nucleotide is a 2'-O-(2-methoxyethyl)(MOE) modified nucleotide. In certain embodiments, 2'-O-(MOE) modified nucleotides are arranged in a gapmer motif, e.g., a 5-9-4 gapmer format, where nine non-2'-O-(MOE) modified nucleotides are adjacent to four or five 2'-O-(MOE) modified nucleotides on one or both sides. In certain embodiments, antisense compounds targeting EXOC2, Ku80, or Task1 nucleic acids include a sterically barren chemical modification format. In some embodiments of the sterically barren chemical modification format, all nucleotides in the antisense compound are 2'-O-(2-methoxyethyl)(MOE) modified nucleotides. In some embodiments of the sterically barren chemical modification format, all nucleotides in the antisense compound are tricyclo-DNA modified nucleotides. In some embodiments of the sterically barren chemical modification format, the antisense compound includes at least one MOE modified nucleotide and at least one tricyclo-DNA modified nucleotide. Many different chemical modification patterns, sterically barren antisense oligonucleotides, are conceivable.For example, but not limited to, sterically blocked antisense oligonucleotides may include mixtures of different types of modifications, such as 2'-O-(2-methoxyethyl) modifications, LNA modifications, tricyclo-DNA modifications, and mixtures of DNA modifications where the DNA stretch is four nucleotides or less.

[0179] In some embodiments, the antisense compounds of the Disclosure induce RNase H-mediated cleavage of the EXOC2, Ku80, or Task1 transcript. In such embodiments, the antisense compounds may be referred to as RNase H-dependent antisense compounds. In some embodiments, the antisense compounds are RNase H-dependent antisense oligonucleotides. In some embodiments, the antisense oligonucleotides of the Disclosure are RNase H-dependent antisense oligonucleotides and may be single-stranded chemically modified oligonucleotides that bind to a complementary sequence in a target transcript (e.g., the EXOC2, Ku80, or Task1 transcript). The RNase H-dependent antisense oligonucleotides of the Disclosure reduce the expression of a target gene by RNase H-mediated cleavage of the target transcript and by inhibition of translation by ribosome steric blockade. In some embodiments, the antisense compounds of the present disclosure can mediate the cleavage of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more of the EXOC2, Ku80, or Task1 transcript by RNase-H. In one embodiment, the antisense compound can mediate the cleavage of at least 80% of the EXOC2, Ku80, or Task1 transcript by RNase-H. In one embodiment, the antisense compound can mediate the cleavage of at least 90% of the EXOC2, Ku80, or Task1 transcript by RNase-H.

[0180] In one particular embodiment, the antisense compound targeting the EXOC2, Ku80, or Task1 transcript has a length of approximately 6 to approximately 24 subunits. In other embodiments, the antisense compound targeting the EXOC2, Ku80, or Task1 transcript has a length of approximately 8 to approximately 80 subunits. For example, an antisense compound is a range defined by 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, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits, or any two of the above values. In some embodiments, the antisense compound is a linked subunit with a length of less than 40 units. In some embodiments, the antisense compound is a linked subunit with a length of about 10 to about 30 units. In some embodiments, the antisense compound is a linked subunit with a length of about 12 to about 25 units. In some embodiments, the antisense compound is a linked subunit with a length of about 15 to about 20 units. In some embodiments, the antisense compound is an antisense oligonucleotide targeting EXOC2, Ku80, or Task1 transcript, and the linked subunit is a linked nucleotide.

[0181] In certain embodiments, antisense compounds targeting EXOC2, Ku80, or Task1 transcripts can be shortened or cleaved. For example, a single subunit can be deleted from the 5' end (5' truncation) or from the 3' end (3' truncation). For shortened or cleaved antisense compounds targeting EXOC2, Ku80, or Task1 transcripts, two subunits can be deleted from the 5' end of the antisense compound, or two subunits can be deleted from the 3' end. Alternatively, in an antisense compound where, for example, one nucleotide is deleted from the 5' end and one nucleoside is deleted from the 3' end, the deleted nucleosides can be dispersed throughout the antisense compound.

[0182] If a single additional subunit is present in an extended antisense compound, the additional subunit can be located at the 5' or 3' end of the antisense compound. If two or more additional subunits are present, for example, in an antisense compound where two subunits are added to the 5' end (5'-addition) or, alternatively, to the 3' end (3'-addition), the added subunits can be adjacent to each other. Alternatively, for example, in an antisense compound where one subunit is added to the 5' end and one subunit is added to the 3' end, the added subunits can be dispersed throughout the antisense compound.

[0183] Without eliminating activity, the length of antisense compounds such as antisense oligonucleotides can be increased or decreased, and / or mismatched bases can be introduced. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides with lengths of 13 to 25 nucleic acid bases were tested in an oocyte injection model for their ability to induce cleavage of target RNA. Antisense oligonucleotides with lengths of 25 nucleic acid bases containing 8 or 11 mismatched bases near the end were able to induce specific cleavage of target mRNA, although not as effectively as antisense oligonucleotides without mismatches. Similarly, target-specific cleavage was achieved using 13-nucleotide antisense oligonucleotides (including those with one or three mismatches).

[0184] In a particular embodiment, the antisense oligonucleotide is expressed as: Including ABC, in the formula, A contains approximately 0 to 18 modified nucleotides. B contains approximately 0 to approximately 4 deoxyribonucleic acid (DNA) nucleotides and / or DNA-like nucleotides, and C contains approximately 0 to 18 modified nucleotides. The total length of an antisense oligonucleotide is approximately 10 to 30 nucleotides.

[0185] Antisense oligonucleotides containing four or fewer DNA and / or DNA-like nucleotides in "B" should not recruit RNase H to induce target cleavage. In these cases, the antisense oligonucleotide can act as a steric barrier rather than a gapmer format.

[0186] Branched antisense compounds The disclosure also provides branched antisense compounds comprising two or more target recognition sequences that target a portion of EXOC2, Ku80, or Task1 nucleic acid. The branched antisense compounds of the disclosure may be, for example, branched antisense oligonucleotide compounds.

[0187] As used herein, the terms “branched antisense compound” or “branched antisense oligonucleotide” refer to two or more antisense compounds or antisense oligonucleotides that are connected to one another.

[0188] In one embodiment, the branched oligonucleotide compound comprises two or more target recognition sequences, which are linked to each other by one or more portions selected from linkers, spacers, and branch points. The target recognition sequences are described herein. In some embodiments, the branched oligonucleotide compound comprises two, three, four, five, six, seven, eight or more target recognition sequences, each of which comprises a 5' end and a 3' end, and each target recognition sequence is independently linked to a linker, a spacer, or a branch point at the 5' or 3' end. In some embodiments, each target recognition sequence is linked to a linker, a spacer, or a branch point at the 5' end. In some embodiments, each target recognition sequence is linked to a linker, a spacer, or a branch point at the 3' end. In another embodiment, each target recognition sequence is linked to a linker, a spacer, or a branch point. In some embodiments, each of the target recognition sequences is an antisense compound and / or oligonucleotide targeting a portion of EXOC2, Ku80, or Task1 nucleic acid.

[0189] In some embodiments, the branched oligonucleotide compounds of the present disclosure are of the formula L-(N) n It has, In the formula, N represents the target recognition sequence of the present disclosure, n represents an integer, for example, 2, 3, 4, 5, 6, 7, or 8, and L represents a linker selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, phosphoric acid, phosphonic acid, phosphoramidate, ester, amide, triazole, and any combination thereof.

[0190] In some embodiments, the branched oligonucleotide compound of the present disclosure has the formula L-(N) n and has In the formula, the compound optionally further comprises one or more branch points B, and the compound optionally further comprises one or more spacers S. In such embodiments, each of the one or more branch points B independently represents a polyvalent organic species or a derivative thereof, and each of the one or more spacers S independently is selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, phosphoric acid, phosphonic acid, phosphoramidate, ester, amide, triazole, and any combination thereof. In some embodiments, the spacer S is biodegradable. For example, the spacer S can include a moiety that is susceptible to cleavage by a nuclease, a protease, a change in pH, or reduction or oxidation. In such embodiments, the spacer can include a peptide, a phosphodiester-linked nucleotide, a disulfide bond, a pH-sensitive linkage, or other biodegradable moiety. For example, the branched oligonucleotide compound of the present disclosure having the formula L-(N)n can have, but is not limited to, is not limited to any form, for example,

Chemical formula

Chemical formula

[0191] Target recognition sequence This disclosure provides antisense oligonucleotides comprising a target recognition sequence that targets a portion of EXOC2, Ku80, or Task1 nucleic acid. In certain embodiments, the antisense oligonucleotide has a nucleic acid base sequence that, when written in the 5' to 3' direction, comprises the reverse complement of a portion of EXOC2, Ku80, or Task1 nucleic acid. In certain such embodiments, the antisense oligonucleotide has a nucleic acid base sequence that, when written in the 5' to 3' direction, comprises the reverse complement of a portion of EXOC2, Ku80, or Task1 nucleic acid.

[0192] In certain embodiments, the target region is a structurally defined region of the EXOC2, Ku80, or Task1 nucleic acid. For example, the target region can encompass a 3' untranslated region (UTR), a 5' untranslated region (UTR), an exon, an intron, an exon / intron junction, a coding region, a translation start region, a translation end region, or other defined nucleic acid regions, such as an open reading frame, or a junction between an open reading frame and an untranslated region, and any combination thereof. The structurally defined regions of EXOC2, Ku80, and Task1 can be obtained by deposit number from a sequence database such as NCBI, and such information is incorporated herein by reference. In certain embodiments, the target region can encompass the sequence from the 5' target site of one target segment within the target region to the 3' target site of another target segment within the same target region.

[0193] Targeting involves determining at least one target segment into which the antisense oligonucleotide hybridizes to produce the desired effect. In certain embodiments, the desired effect is an increase in the level of the transcript target nucleic acid, i.e., an increase in the level of EXOC2, Ku80, or Task1 transcript. In certain embodiments, the desired effect is an increase in the level of the protein encoded by the target nucleic acid, or a phenotypic change associated with the target nucleic acid, e.g., an increase in the level of the EXOC2, Ku80, or Task1 protein.

[0194] A target region may contain one or more target segments. Multiple target segments within a target region may overlap. Alternatively, they may not overlap. In certain embodiments, target segments within a target region are separated by approximately 300 or fewer nucleotides. In certain embodiments, target segments within a target region are separated by multiple nucleotides, each in a range defined by approximately 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or any two of the aforementioned values. In certain embodiments, target segments within a target region are separated by 5 or fewer nucleotides on the target nucleic acid. In certain embodiments, target segments are contiguous.

[0195] Suitable target segments can be found within the 5'UTR, coding region, 3'UTR, introns, exons, and / or exon / intron junctions. Target segments containing start or stop codons are also suitable target segments. Suitable target segments can exclude certain structurally defined regions, such as start or stop codons. In certain embodiments, the target segment is found in intron 1 of the EXOC2, Ku80, and Task1 genes. In certain embodiments, the target segment is found upstream (5') of the nucleotide repeat region in intron 1 of the EXOC2, Ku80, and Task1 genes. In certain embodiments, the target segment is found downstream (3') of the nucleotide repeat region in intron 1 of the EXOC2, Ku80, and Task1 genes. In certain embodiments, the target segment is found upstream or downstream of a nucleotide repeat region in intron 1 of the EXOC2, Ku80, and Task1 genes, and is unique to the human genome (i.e., the target segment nucleic acid sequence is found only once in the human genome).

[0196] Determining an appropriate target segment may involve comparing the sequences of the target nucleic acids (e.g., EXOC2, Ku80, and Task1) with other sequences across the entire genome. For example, the BLAST algorithm can be used to identify regions of similarity between different nucleic acids. This comparison can prevent the selection of antisense oligonucleotide sequences that can non-specifically hybridize to sequences other than the selected target nucleic acids (i.e., non-target or off-target sequences). Determining an appropriate target segment may involve comparing the sequences of the target nucleic acids (e.g., EXOC2, Ku80, and Task1 transcripts) across several species. For example, various sequence alignment software can be used to identify regions of similar or identical sequences across species. In a particular embodiment, the EXOC2, Ku80, and Task1 transcript target segment nucleic acid sequences are unique to the human genome (i.e., the target segment nucleic acid sequences are found only once in the human genome).

[0197] Antisense oligonucleotides and target nucleic acids (e.g., EXOC2, Ku80, and Task1 transcripts or parts thereof) are complementary to each other if a sufficient number of nucleic acid bases of the antisense oligonucleotide can form hydrogen bonds with the corresponding nucleic acid bases of the target nucleic acid to produce the desired effect (e.g., increased expression of target nucleic acids such as EXOC2, Ku80, and Task1 transcripts or parts thereof).

[0198] Non-complementary nucleic acid bases between the antisense oligonucleotide and the EXOC2, Ku80, and Task1 nucleic acids are acceptable, as long as the antisense oligonucleotide can still hybridize specifically to the target nucleic acid. Furthermore, the antisense oligonucleotide can hybridize onto one or more segments of the EXOC2, Ku80, and Task1 nucleic acids in such a way that intervening or adjacent segments do not participate in the hybridization event (e.g., loop structure, mismatch, or hairpin structure).

[0199] In certain embodiments, the antisense oligonucleotides or any particular portion thereof provided herein are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to EXOC2, Ku80, and Task1 nucleic acids, target regions, target segments, or any particular portion thereof.

[0200] For example, an antisense oligonucleotide in which 18 of the 20 nucleic acid bases are complementary to the target region (e.g., the isolength portions of EXOC2, Ku80, and Task1 transcripts), and thus specifically hybridizes, represents 90 percent complementarity. In this example, the remaining non-complementary nucleic acid bases may be clustered, or complementary nucleic acid bases may be scattered among the remaining non-complementary nucleic acid bases, and they do not need to be consecutive with each other or with complementary nucleic acid bases. Thus, an antisense oligonucleotide of 18 nucleic acid bases having two regions that are perfectly complementary to the target nucleic acid and four (quad) non-complementary nucleic acid bases adjacent to it has 77.8% total complementarity to the target nucleic acid and is therefore included in the scope of this disclosure. The complementarity percentage of antisense oligonucleotides to a target nucleic acid region can be routinely determined using the BLAST program (basic local alignment search tool) and the PowerBLAST program, which is known in the art (Altschul et al., J.Mol.Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). Homology percentages, sequence identity percentages, or sequence complementarity percentages can be determined using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) with default settings, for example, using the Smith and Waterman algorithm (Adv.Appl.Math., 1981, 2, 482-489).

[0201] In certain embodiments, the antisense oligonucleotide or a particular portion thereof provided herein is perfectly complementary (i.e., 100% complementary) to the target nucleic acid or a particular portion thereof. For example, an antisense oligonucleotide can be perfectly complementary to EXOC2, Ku80, or Task1 nucleic acid, or to a target region, or to a target segment or its target sequence. As used herein, “perfectly complementary” means that each nucleic acid base of the antisense oligonucleotide can precisely base-pair with the corresponding nucleic acid base of the target nucleic acid. For example, a 20-nucleotide antisense oligonucleotide is perfectly complementary to a 400-nucleotide target sequence, insofar as there is a corresponding 20-nucleotide portion of the target nucleic acid that is perfectly complementary to the antisense oligonucleotide. Perfectly complementary can also be used in reference to a particular portion of a first and / or second nucleic acid. For example, a 20-nucleotide portion of a 30-nucleotide antisense oligonucleotide can be “perfectly complementary” to a 400-nucleotide target sequence. The 20-nucleotide portion of a 30-nucleotide oligonucleotide is perfectly complementary to the target sequence if the target sequence has a corresponding 20-nucleotide portion (where each nucleotide is complementary to the 20-nucleotide portion of the antisense oligonucleotide). At the same time, the entire 30-nucleotide antisense oligonucleotide may or may not be perfectly complementary to the target sequence, depending on whether the remaining 10 nucleotides of the antisense oligonucleotide are also complementary to the target sequence.

[0202] In one embodiment, the disclosure provides an antisense oligonucleotide comprising regions complementary to introns of EXOC2, Ku80, and Task1 transcripts, wherein the antisense oligonucleotide does not contain regions complementary to other sites in the human genome (i.e., the target sequence of the antisense oligonucleotide is unique in the human genome).

[0203] In another aspect, the present disclosure provides an antisense oligonucleotide comprising regions of complementarity to EXOC2, Ku80, and intron 1 of the Task1 transcript, wherein the antisense oligonucleotide does not comprise regions of complementarity to other sites in the human genome (i.e., the target sequence of the antisense oligonucleotide is unique in the human genome).

[0204] In certain embodiments, the antisense oligonucleotide comprises [X S ] a [ X S b [[ID=...]] S X] c a sequence modification pattern of, wherein "a" represents an integer from 0 to 8, "b" represents an integer from 6 to 18, "c" represents an integer from 0 to 8, "s" represents a phosphorothioate internucleoside linkage, 「X」 is adenosine, guanosine, cytidine, thymine, or uracil, X comprises a 2'-O-(2-methoxyethyl) modification or a tricyclo-DNA modification (i.e., each of adenosine, guanosine, cytidine, thymine, and uracil comprises a 2'-O-(2-methoxyethyl) modification or a tricyclo-DNA modification), "X" is adenosine, guanosine, cytidine, thymine, or uracil, and X comprises a 2'-deoxy modification (i.e., each of adenosine, guanosine, cytidine, thymine, and uracil comprises a 2'-deoxy modification), the sum of a, b, and c is 12 or more.

[0205] In certain embodiments, the antisense oligonucleotide comprises X S X S X ​​S X S X S X S X S X S X S X S X S X S X S X S X S X S X S X including the array modification pattern of, wherein, "s" represents a phosphorothioate nucleoside internucleoside linkage, 「X」 is adenosine, guanosine, cytidine, thymine, or uracil, X includes a 2'-O-(2-methoxyethyl) modification or a tricyclo-DNA modification, "X" is adenosine, guanosine, cytidine, thymine, or uracil, and X includes a 2'-deoxy modification.

[0206] In certain embodiments, the antisense oligonucleotide X S X S X S X S X S X S X S X S X S X S X S X S X S X S X S X S X S X including the array modification pattern of X, wherein, "s" represents a linkage between phosphorothioate nucleosides. 「X」 These are adenosine, guanosine, cytidine, thymine, or uracil. X is , including 2'-O-(2-methoxyethyl) modification, "X" is adenosine, guanosine, cytidine, thymine, or uracil, and X contains a 2'-deoxy modification.

[0207] In a particular embodiment, the antisense oligonucleotide is [X S ] d [ S X] e The formula includes the array modification pattern, "d" represents an integer from 0 to 40. "e" represents an integer from 0 to 40. "s" represents a linkage between phosphorothioate nucleosides. 「X」 These are adenosine, guanosine, cytidine, thymine, or uracil. X This includes 2'-O-(2-methoxyethyl) modification or tricyclo-DNA modification, The sum of d and e is 10 or more.

[0208] In a particular embodiment, the antisense oligonucleotide is X S X S X S X S X S X S X S X S X S X S X SX S X S X S X S X S X S X The formula includes the array modification pattern, "s" represents a linkage between phosphorothioate nucleosides. 「X」 These are adenosine, guanosine, cytidine, thymine, or uracil. X This includes 2'-O-(2-methoxyethyl) modification or tricyclo-DNA modification.

[0209] In a particular embodiment, the antisense oligonucleotide is X S X S X S X S X S X S X S X S X S X S X S X S X S X S X S X S X S X The formula includes the array modification pattern, "s" represents a linkage between phosphorothioate nucleosides. 「X」 These are adenosine, guanosine, cytidine, thymine, or uracil. X is, including 2'-O-(2-methoxyethyl) modification.

[0210] In a particular embodiment, each cytosine is 5-methylcytosine.

[0211] In one embodiment, the present disclosure provides an antisense oligonucleotide comprising the following sequence:

[0212] G S C S A S A S U S A S C S A S T S G S G S A S T S T S G S G S G S G , in the formula, "s" represents a linkage between phosphorothioate nucleosides. 「A」 This is adenosine containing a 2'-O-(2-methoxyethyl) modification, 「G」 This is guanosine containing a 2'-O-(2-methoxyethyl) modification, 「C」 This is a cytidine containing a 2'-O-(2-methoxyethyl) modification, 「U」 This is a thymine containing a 2'-O-(2-methoxyethyl) modification, "A" is adenosine with a 2'-deoxy modification, "G" is guanosine with a 2'-deoxy modification, "C" is a cytidine containing a 2'-deoxy modification. "T" is a thymine containing a 2'-deoxy modification.

[0213] In one embodiment, the present disclosure provides an antisense oligonucleotide comprising the following sequence:

[0214] G S C S A S A S U S A S C S A S U S G S G S A S U S U S G S G S G S G , during the ceremony "s" represents a linkage between phosphorothioate nucleosides. 「A」 This is adenosine containing a 2'-O-(2-methoxyethyl) modification, 「G」 This is guanosine containing a 2'-O-(2-methoxyethyl) modification, 「C」 This is a cytidine containing a 2'-O-(2-methoxyethyl) modification, 「U」 This is a thymine containing a 2'-O-(2-methoxyethyl) modification.

[0215] In one embodiment, the present disclosure provides an antisense oligonucleotide comprising the following sequence:

[0216] G S US A S C S A S A S A S C S U S C S C S G S G S A S G S A S G S C , in the formula, "s" represents a linkage between phosphorothioate nucleosides. 「A」 This is adenosine containing a 2'-O-(2-methoxyethyl) modification, 「G」 This is guanosine containing a 2'-O-(2-methoxyethyl) modification, 「C」 This is a cytidine containing a 2'-O-(2-methoxyethyl) modification, 「U」 This is a thymine containing a 2'-O-(2-methoxyethyl) modification.

[0217] In certain embodiments, two or more antisense oligonucleotides are linked to each other via a linker. In certain embodiments, the linker is a cleavable linker. In certain embodiments, the cleavable linker degrades when cleaved. In certain embodiments, the cleavable linker is a nuclease-cleavable linker containing a phosphodiester linkage. In certain embodiments, the nuclease-cleavable linker contains about 2 to about 8 nucleotides in length. In certain embodiments, the nuclease-cleavable linker contains about 6 nucleotides in length. In certain embodiments, the cleavable linker is cleaved under reducing conditions or under changing pH conditions. In certain embodiments, the cleavable linker is cleaved by an intracellular nuclease or endosomal nuclease. In certain embodiments, the cleavable linker is cleaved by an intracellular protease or endosomal protease.

[0218] Conjugated flat sense oligonucleotides Antisense oligonucleotides can be covalently linked to one or more moieties, ligands, sequences, or conjugates that enhance the activity, cell distribution, or cell uptake of the resulting antisense oligonucleotide. Antisense oligonucleotides can also be covalently linked to one or more moieties, ligands, sequences, or conjugates that enhance and / or optimize pharmacokinetic parameters. Various pharmacokinetic parameters include absorbance, concentration of the compound in the body, degree to which the compound penetrates the body, rate of compound elimination / clearance, and volume of plasma from which the compound is removed per unit time.

[0219] The conjugated group may include a lysosomal ligand or sequence. In some embodiments, the lysosomal sequence is a poly-G sequence. In some embodiments, the poly-G sequence is a 2-30 poly-G sequence. In some embodiments, the poly-G sequence is a 2, 3, 4, 5, 6, 7, 8, 9, or 10 poly-G sequence.

[0220] The conjugated group may include a hydrophobic moiety. In certain embodiments, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides and nucleoside analogs, endocannabinoids, and vitamins. In certain embodiments, the steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA). In certain embodiments, the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanic acid (DCA). In certain embodiments, the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, and their derivatives or metabolites. In certain embodiments, the vitamin is selected from the group consisting of retinoic acid and alpha-succinate tocopherol.

[0221] In certain embodiments, the antisense compounds of this disclosure are conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand containing a cationic group. In certain embodiments, the lipophilic moiety is selected from the group consisting of cholesterol, vitamin E, vitamin K, vitamin A, folic acid, or a cationic dye (e.g., Cy3). In an exemplary embodiment, the lipophilic moiety is cholesterol. Other lipophilic moieties include cholic acid, adamantane acetate, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. Diverse lipid conjugates can preferentially drive oligonucleotide uptake into different tissues (Biscans et al, Nucleic Acids Res. 2019, 47, 1082-1096).

[0222] Additional conjugated groups include carbohydrates, phospholipids, antibodies, peptides, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. In some embodiments, the conjugation of ligands to antisense oligonucleotides enables recognition by cell surface receptors (see, for example, Wolfrum et al., Nat. Biotechnol. 2007, 25:1149-1157, Hostetler et al., Antiviral Chem. Chemother. 2001, 12:61-70, and Prakash et al., Nucleic Acids Res. 2014, 42:8796-807). In certain embodiments, the conjugate is a fibronectin type III (FN3) domain, e.g., a senin protein (see, e.g., Goldberg et al., Protein Eng Des Sel. 2016, 29(12):563-572). Various parts, ligands, sequences, or conjugates of this disclosure and means for conjugating them to antisense compounds are described in further detail in WO2017 / 030973A1 and WO2018 / 031933A2, which are incorporated herein by reference.

[0223] Antisense oligonucleotides can also be modified to have one or more stabilizing groups attached to one or both ends of the antisense oligonucleotide to enhance properties such as nuclease stability. These stabilizing groups include cap structures. These terminal modifications can protect antisense oligonucleotides having terminal nucleic acids obtained from exonuclease degradation and aid in intracellular delivery and / or localization. The caps may be present at the 5' end (5' cap) or the 3' end (3' cap), or at both ends. Cap structures include, for example, inverted deoxydecay caps. Further 3' and 5'-stabilizing groups that can be used to cap one or both ends of antisense oligonucleotides to confer nuclease stability include those disclosed in WO03 / 004602, published January 16, 2003.

[0224] In some embodiments, the antisense oligonucleotides of the Disclosure include a conjugate. In one embodiment, the antisense oligonucleotides of the Disclosure include an antisense oligonucleotide sequence and a conjugate, the conjugate being linked to the antisense oligonucleotide sequence. In some embodiments, the conjugate is selected from any of the conjugates described herein, e.g., hydrophobic conjugates, tissue-targeting conjugates, or conjugates designed to optimize pharmacokinetic parameters. Hydrophobic conjugates useful for conjugating the antisense oligonucleotides of the Disclosure include hexadecyloxypropyl conjugates, cholesterol conjugates, polyunsaturated fatty acid conjugates, and other conjugates known in the art that can improve the cellular uptake of the conjugated antisense oligonucleotide. In some embodiments, the conjugate can be a tissue-targeting conjugate, e.g., a carbohydrate conjugate or a peptide conjugate, or any conjugate known in the art that can target the antisense oligonucleotide of the Disclosure to a specific tissue. In some embodiments, the antisense oligonucleotides of the Disclosure are conjugated with polyethylene glycol conjugates. In one embodiment, polyethylene glycol-conjugated antisense oligonucleotides optimize the pharmacokinetic properties of the antisense oligonucleotide.

[0225] In some embodiments, this disclosure provides biocleavable analogues of the antisense oligonucleotides described herein. In such cases, the biocleavable analogues include hydrophobic conjugates that result in stronger association with the cell membrane and the linker. In one embodiment, the linker is a cleavable linker that, when cleaved, releases the antisense oligonucleotide, for example, to an endosome. In some embodiments, the antisense compound comprises a cleavable linker that degrades when cleaved. In some embodiments, the linker is a nuclease-cleavable linker containing a phosphodiester linkage. In some embodiments, the nuclease-cleavable linker containing a phosphodiester linkage has a length of about 2 to about 8 nucleotides. For example, the nuclease-cleavable phosphodiester linker can have a length of 3, 4, 5, 6, 7, 8 nucleotides or more, for example, a length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 nucleotides or more. In one embodiment, the nuclease-cleavable linker contains approximately six nucleotides in length. In one embodiment, the cleavable linker is cleaved after intracellularization. In one embodiment, the cleavable linker is cleaved within endosomes. In one embodiment, the cleavable linker is cleaved under reducing conditions. In some embodiments, the cleavable linker is cleaved under changing pH conditions, for example, when the pH decreases or increases. In one embodiment, the cleavable linker is cleaved by an intracellular nuclease or protease. In one embodiment, the cleavable linker is cleaved by an endosomal nuclease or protease.

[0226] Pharmaceutical compositions and preparations This specification provides pharmaceutical compositions and formulations comprising antisense compounds described herein. For example, antisense oligonucleotides described herein can be mixed, encapsulated, conjugated, or separately associated with other molecules, molecular structures, or mixtures of compounds. The pharmaceutical compositions of this disclosure are formulated to suit their intended route of administration. Examples of central nervous system administration routes include intrathecal, intraventricular, or intrastriatal administration. In some embodiments, administration may be performed using implantable devices such as an Omaya reservoir or an implantable intrathecal catheter. Examples of systemic administration include intravenous, subcutaneous, or intramuscular administration. The route of administration is partially determined by the target tissue of the antisense compound. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents, e.g., water for injection, saline solution, Ringer's lactate solution, Elliotts B solution, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetates, citrates, carbonates, or phosphates; and agents for adjusting tonicity, e.g., sodium chloride or dextrose. pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Pharmaceutical compositions may be enclosed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0227] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for preparing sterile injection solutions or dispersions immediately before use. In all cases, the composition must be sterile and fluid enough to be easily injected. The composition must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is appropriate to include isotonic agents, such as sugar and polyalcohols (mannitol, sorbitol, sodium chloride, etc.) in the composition. Sustained absorption of an injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0228] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound, along with one or a combination of the components listed above as needed, into a suitable solvent, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, some methods of preparation include vacuum drying and freeze-drying, from which a powder is obtained by combining the active ingredient and any desired additional components from a pre-sterilized filtered solution.

[0229] The pharmaceutical compositions and formulations provided herein can, in some embodiments, be conveniently presented in unit dosage forms and can be prepared according to the prior art. Such techniques may include associating the active ingredient with a pharmaceutical carrier(s) or excipient(s). Generally, formulations can be prepared by homogeneously and closely associating the active ingredient with a liquid carrier, a micronized solid carrier, or both, and then, if necessary, shaping the product (e.g., to a specific particle size for delivery). In one embodiment, the pharmaceutical formulation is prepared for intrathecal, intraventricular, or striatal administration in a suitable solvent, e.g., water or saline. In another embodiment, the pharmaceutical formulation is prepared for intravenous, subcutaneous, or intramuscular administration in a suitable solvent, e.g., water or saline.

[0230] The agents of this disclosure, for example, antisense compounds targeting EXOC2, Ku80, and Task1 transcripts, can also be administered by transfection or infection using methods known in the art, including, but not limited to, those described in McCaffrey et al. (2002), Nature, 418(6893), 38-9 (hydrodynamic transfection), Xia et al. (2002), Nature Biotechnol., 20(10), 1006-10 (viral-mediated delivery), or Putnam (1996), Am.J. Health Syst. Pharm. 53(2), 151-160, erratum at Am.J. Health Syst. Pharm. 53(3), 325 (1996).

[0231] The agents of this disclosure, such as antisense compounds targeting EXOC2, Ku80, and Task1 transcripts, can also be administered by any method suitable for the administration of nucleic acid agents, such as DNA vaccines. These methods include needle-free methods such as gene guns, bioinjectors, skin patches, and the particulate DNA vaccine technology disclosed in U.S. Patent No. 6,194,389, as well as percutaneous needle-free vaccination of mammals using powdered vaccines disclosed in U.S. Patent No. 6,168,587. In particular, intranasal delivery is possible, as described in Hamajima et al. (1998), Clin. Immunol. Immunopathol., 88(2), 205-10, etc. Liposomes (e.g., described in U.S. Patent No. 6,472,375) and microencapsulation can also be used. Biodegradable, targetable particulate delivery systems can also be used (e.g., described in U.S. Patent No. 6,471,996).

[0232] In one embodiment, the activator is prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled-release formulation comprising an implant and a microencapsulation delivery system. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid can be used. Methods for preparing such formulations are apparent to those skilled in the art. The materials can also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes targeting infected cells and containing monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, the method described in U.S. Patent No. 4,522,811.

[0233] The toxicity and therapeutic effects of such compounds can be determined, for example, by standard pharmaceutical procedures in cell cultures or experimental animals to determine the LD50 (lethal dose for 50% of the population) and ED50 (therapeutably effective dose for 50% of the population). The dose-to-toxicity ratio is the therapeutic index and can be expressed as the LD50 / ED50 ratio. Compounds exhibiting a large therapeutic index are suitable. While compounds exhibiting toxic side effects can be used, care must be taken to design a delivery system that directs such compounds to the site of the affected tissue in order to minimize potential damage to uninfected cells and thereby reduce side effects.

[0234] Data obtained from cell culture assays and animal studies can be used when formulating dose ranges for use in humans. Doses of such compounds are within a circulating concentration range that includes an ED50 with little to no toxicity. Doses can vary within this range depending on the form of administration used and the route of administration utilized. For any compound used in the methods of this disclosure, the therapeutically effective dose can first be estimated from a cell culture assay. The dose can be formulated in animal models to achieve a circulating plasma concentration range that includes an EC50 (i.e., the concentration of the test compound that achieves half of the maximum response) determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0235] The pharmaceutical composition may be contained within a container, pack, or dispenser, along with instructions for administration.

[0236] Antisense compounds targeting EXOC2, Ku80, and Task1 nucleic acids can be used in pharmaceutical compositions by combining the antisense compounds with a suitable pharmaceutically acceptable diluent or carrier. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS). PBS is a suitable diluent for use in compositions delivered parenterally. Therefore, in one embodiment, the method described herein uses a pharmaceutical composition comprising an antisense compound targeting EXOC2, Ku80, and Task1 nucleic acids and a pharmaceutically acceptable diluent. In a particular embodiment, the pharmaceutically acceptable diluent is PBS. In a particular embodiment, the antisense compound is an antisense oligonucleotide.

[0237] In certain embodiments, the pharmaceutically acceptable diluent is designed to mimic the composition of cerebrospinal fluid. Therefore, Mg 2+ and Ca 2+ It may contain divalent salts such as those listed above. Elliott B solution is a suitable diluent for use in compositions delivered to cerebrospinal fluid. Those skilled in the art will understand that other buffer solutions with varying concentrations of different monovalent and divalent ions may also be suitable as pharmaceutically acceptable diluents.

[0238] A pharmaceutical composition comprising an antisense compound includes any pharmaceutically acceptable salt of such ester, ester or salt of such ester, or any other oligonucleotide, which can provide (directly or indirectly) a biologically active metabolite or residue thereof when administered to an animal, including a human. Therefore, for example, this disclosure covers pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other biological equivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. A prodrug may involve the incorporation of further nucleosides at one or both ends of the antisense compound, which are cleaved in the body by endogenous nucleases to form an active antisense compound.

[0239] Treatment method This disclosure provides a method for treating subjects having EXOC2, Ku80, or Task1-related disorders. The treatment method comprises the step of administering an effective amount of the antisense compound described herein to a subject in need of treatment. In certain embodiments, the antisense compound comprises a target recognition sequence that is sufficiently complementary to the EXOC2, Ku80, or Task1 nucleic acid (e.g., an EXOC2, Ku80, or Task1 transcript) in order to induce cleavage of the EXOC2, Ku80, or Task1 nucleic acid by RNase H. In certain embodiments, the antisense compound comprises a target recognition sequence that is sufficiently complementary to the EXOC2, Ku80, or Task1 nucleic acid (e.g., an EXOC2, Ku80, or Task1 transcript) in order to increase the expression of the EXOC2, Ku80, or Task1 nucleic acid.

[0240] A method for treating subjects with EXOC2, Ku80, or Task1-related disorders is useful for treating any EXOC2, Ku80, or Task1-related disorders known to those skilled in the art.

[0241] In another aspect, the disclosure provides a method for treating or managing amyotrophic lateral sclerosis (ALS) in a patient, the method comprising the step of administering to the patient a therapeutically effective amount of an oligonucleotide complementary to SOD1 as described herein.

[0242] In another aspect, the disclosure provides a method for treating or managing primary hyperlipidemia in a patient, the method comprising the step of administering a therapeutically effective dose of a PCSK9-targeting dsRNA as described herein to the patient.

[0243] In some embodiments, primary hyperlipidemia is heterozygous familial hypercholesterolemia (HeFH).

[0244] In another aspect, the present disclosure provides a method for reducing low-density lipoprotein cholesterol (LDL-C) in a patient, the method comprising the step of administering a therapeutically effective dose of a PCSK9-targeting dsRNA as described herein to the patient.

[0245] The content of any papers, patents, and patent applications, as well as all other documents and electronically available information, mentioned or cited herein are incorporated herein by reference in whole to the same extent as when each individual publication specifically and individually indicates that it is incorporated by reference. The applicant reserves the right to physically incorporate into this application any material and information from any such papers, patents, patent applications, or other physical and electronic documents.

[0246] While this disclosure has been described in relation to its specific embodiments, those skilled in the art should understand that various modifications can be made and equivalents can be substituted without departing from the true spirit and scope of this disclosure. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein can be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications can be made to adapt specific circumstances, materials, compositions, processes, or treatment steps to the purpose, spirit, and scope of this disclosure. All such modifications are intended to fall within the scope of the claims appended herein. While specific embodiments have been described in detail so far, embodiments will be better understood by referring to the following examples. These examples are included for illustrative purposes only and are not intended to limit the scope. [Examples]

[0247] The present disclosure is further illustrated by the following embodiments, but this should not be construed as a further limitation.

[0248] Example 1 - Materials and Method iPSC culture iPSC strains from the C9ORF72 carrier and its isogenic control were previously characterized (Lopez-Gonzalez et al., 2019). iPSCs were cultured (Lopez-Gonzalez et al., 2019). Briefly, iPSCs were maintained in 6-well plates coated with Matrigel (Catalog No. 354230, Corning) diluted 1:100 in Knock Out DMEM / F-12 (Catalog No. 12660012, Gibco) in mTeSR1 medium (mTeSR1 basal medium with mTeSR1 5X supplement, Stem Cell Technologies). The mTeSR1 medium was changed daily. iPSCs were passaged every 4–6 days. Cells were washed with DPBS, dissociated with Accutase ((catalog number SCR005, Millipore) diluted 1:2 with DPBS) at room temperature (RT) for approximately 1 minute, washed with DPBS again, and scraped off in fresh mTeSR1 medium using a cell lifter. After reaching the desired colony size, cells were seeded into Matrigel-coated 6-well plates in fresh mTeSR1 medium. Spontaneously differentiated colonies were manually removed before mitotic and motor neuron differentiation.

[0249] Differentiation of motor neurons iPSCs were differentiated into spinal motor neurons according to the protocol from Du et al., 2015, with some modifications (Du et al., 2015, Lopez-Gonzalez et al., 2019). Small-sized iPSCs were seeded in mTeSR1 medium at a concentration of approximately 50% in Matrigel-coated 6-well plates. The following day, the culture medium was replaced with neuroepithelial precursor (NEP) induction medium, supplemented with 3 μM CHIR99021 (catalog no. 72054 Stem Cell Technologies), 2 μM DMH1 (catalog no. 4126 Tocris Bioscience), and 2 μM SB431542 (catalog no. 04-0010-10 Stemgent). The mixture consisted of 1:1 KnockOut DMEM / F-12 medium (catalog no. 12660012 Gibco) and Neurobasal medium (catalog no. 21103049 Gibco), 0.5X N2 supplement (catalog no. 17502-048 Gibco), 0.5X B27 supplement (catalog no. 17504044 Gibco), 0.1 mM ascorbic acid (catalog no. A4403 Sigma), and 1X The drug used was Glutamax (catalog number 35050061, Thermo Fisher Scientific). The NEP medium was changed daily for 6 days. NEP was dissociated with 1:2 diluted Accutase and divided into 1:3 Matrigel-coated 6-well plates. NEP was cultured in motor neuron precursor (MNP) induction medium, and the neuronal medium was supplemented with 1 μM CHIR99021, 2 μM DMH1, 2 μM SB431542, 0.1 μM retinoic acid (catalog number R2625, Sigma), and 0.5 μM purmorphamine (catalog number 540220, Calbichem). The MNP medium was changed daily for 6 days. MNPs were dissociated with a 1:2 dilution of Accutase and cultured for 6 days in a suspension in a 60 mm low-adhesion plate in motor neuron differentiation medium (neuronal medium supplemented with 0.5 μM retinoic acid and 0.1 μM purmorphamine). The medium was changed daily for 6 days.Finally, the neurospheres were dissociated into single cells using Accutase (10 minutes, 37°C) and transferred to motor neuron medium through a 40 μm filter. The neuron medium was supplemented with 0.5 μM retinoic acid, 0.1 μM purmorphamine, 0.1 μM compound E (catalog no. 73954, Stem Cell Technologies), 10 ng / ml BDNF (catalog no. 248-BDB, R&D Systems), 10 ng / ml GDNF (catalog no. PHC7041, Thermo Fisher Scientific), and 1 μg / ml laminin (catalog no. L2020, Sigma). The cells were placed in 5 × 10⁻¹⁶ cells in the motor neuron medium. 5 Cells were seeded at a density of 10 cells / well into Matrigel-coated 12-well plates. After 2 weeks, the neurons were maintained in motor neuron medium free of retinoic acid and purmorphamine. The medium was changed weekly for 3.5 weeks.

[0250] cell culture The HEK293T cell line was purchased from the American Type Culture Collection (ATCC, catalog number CRL-11268). Wild-type (catalog number ab255448) and Lamp2 knockout (catalog number ab255402) cell lines were purchased from Abcam. Cells were maintained in Dulbecco's modified Eagle medium (DMEM, catalog number 10-013-CV, Corning) supplemented with 10% fetal bovine serum (FBS, catalog number 10-437-028, Thermo Fisher Scientific) and 1% penicillin / streptomycin. Cells were divided into 2 × 10⁶ cells for treatment. 5 The cells were seeded in a 12-well plate at a density of one cell per well.

[0251] ASO treatment Three-week-old iPSC-derived neurons or cell lines were treated with ASO at the indicated concentrations for two days. ASO was added directly to the culture medium for free uptake. Cells were harvested two days after ASO treatment for RNA extraction. A list of ASOs is shown in Table 1.

[0252] The non-targeted control (NTC), EXOC2-349, EXOC2-3933, Ku80-624, Ku80-1425, Ku80-2802, Task1-692, and Task1-5968 ASOs were designed by IDT Custom Design Services. All ASOs listed in Table 1 were purchased from IDT.

[0253] Bafilomycin A1 treatment One day after seeding, HEK293T cells were treated with 50 nM bafilomycin A1 (BafA1) or DMSO for 24 hours. The following day, the cells were treated with ASO and 10 nM BafA1. Cells were harvested two days after ASO treatment for RNA extraction.

[0254] RNA extraction and real-time quantitative PCR Total RNA from iPSC-MNs was extracted using the RNeasy Mini kit (catalog number 74106, Qiagen). 1 μg of RNA was reverse transcribed to cDNA using a TaqMan reverse transcription kit (catalog number N8080234, Thermo Fisher Scientific) with a random hexamer. Real-time quantitative PCR was performed using the Applied Biosystems Quant Studio 3 System with SYBR Select Master Mix (catalog number 4472918, Thermo Fisher Scientific). The Ct values ​​of each sample were normalized to cyclophyllin. The relative mRNA levels of each gene were calculated as follows: -ΔΔCt The calculations were performed using the specified method. The qRT-PCR primers are listed in Table 2.

[0255] Example 2 - EXOC2 mRNA levels measured by qRT-PCT analysis for gapmers with and without poly-G ligand, poly-A ligand, or poly-C ligand. Relative EXOC2 mRNA levels were evaluated by qRT-PCT analysis for gapmers with and without poly-G ligand, poly-A ligand, or poly-C ligand (Figure 3).

[0256] Relative EXOC2 mRNA levels were analyzed by qRT-PCR over two days in HEK cells treated with 5uM untargeted control (NTC) or EXOC2 targeting ASOs having gapmer ASO 3933, LysoASO 3933-G5, or other additional sequences 3933-A5 or 3933-C5.

[0257] ASO sequences with poly-G ligands were found to be specific and provide further knockdown compared to ASOs without poly-G ligands. ASO 3933 was found to significantly reduce EXOC2 mRNA levels compared to NTC ASO. The addition of the Lyso sequence (GGGGG) to 3933 and 3933-G5 was found to significantly increase the knockdown efficiency of gapmer ASO 3933. The enhancement of knockdown efficiency was specific to the Lyso sequence, as the addition of (AAAAA) or (CCCCC) to 3933 did not significantly alter relative EXOC2 mRNA levels compared to 3933.

[0258] Example 3 - EXOC2 mRNA levels measured by qRT-PCT analysis for gapmers with and without poly-G ligand of different lengths. Relative EXOC2 mRNA levels were evaluated by qRT-PCT analysis for gapmers with and without poly-G ligands of different lengths (Figure 4).

[0259] Relative EXOC2 mRNA levels were analyzed by qRT-PCR over two days in HEK cells treated with 5uM NTCs containing G3933-G4, 3933-G5, 3933-G10, and 3933-G12 of different lengths, EXOC2-targeted gapmer ASO 3933, and LysoASO.

[0260] ASO sequences containing poly-G ligands were found to have a range of 5 to 10 Gs. ASO 3933 was found to significantly reduce EXOC2 mRNA levels compared to NTC ASO. ASO 3933-G5 and 3933-G10 were found to significantly increase the knockdown efficiency of gapmer ASO 3933. ASO 3933-G4 and 3933-G12 were found not to reduce EXOC2 mRNA levels.

[0261] Example 4 - EXOC2 mRNA level measured by qRT-PCT analysis for EXOC2 349 The relative EXOC2 mRNA levels for EXOC2 349 were evaluated by qRT-PCT analysis (Figure 5).

[0262] Relative EXOC2 mRNA levels were analyzed over two days in iPSC-derived neurons treated with 5 μM NTC, EXOC2 targeting the gapmer ASO 349, LysoASO 349-G10, 349-G7 PS, and other unmodified oligonucleotides 349 no mod, 349 no mod G10, and 349 no mod adjacent G7.

[0263] ASOs with poly-G ligands were found to activate non-functional ASOs in iPSC-derived neurons. Gapmer ASO 349 was found not to alter relative EXOC2 mRNA levels compared to NTC ASO. ASO 349-G10 was found to significantly reduce EXOC2 mRNA levels compared to NTC or ASO 349. ASO 349-G7 PS was found not to alter relative EXOC2 mRNA levels compared to NTC ASO, suggesting that complete or partial PS modification may prevent recognition of the Lyso sequence by the Lamp2C receptor.

[0264] Example 5 - EXOC2 mRNA level measured by qRT-PCT analysis for EXOC2 349 The relative EXOC2 mRNA levels of EXOC2 3933 were evaluated by qRT-PCT analysis (Figure 6).

[0265] Relative EXOC2 mRNA levels in wild-type (WT) and LAMP2 knockout (KO) HeLa cells were analyzed by qRT-PCR after treatment with EXOC2 targeting 5 μM NTC, gapmer ASO 3933, and LysoASO 3933-G5 for 2 days. Gapmer ASO 3933 significantly reduced relative EXOC2 mRNA levels in WT and LAMP2 KO cells compared to NTC ASO.

[0266] The effects of LysoASO were found to be mediated via the LAMP2 receptor. LysoASO 3933-G5 was found to further improve the knockdown efficiency of ASO 3933. EXOC2 mRNA levels were found to be significantly lower in LysoASO 3933-G5 groups compared to ASO 3933 groups in WT cells. However, this effect was not observed in LAMP2 KO cells, and LysoASO 3933-G5 did not alter EXOC2 mRNA levels compared to NTC or ASO 3933.

[0267] Example 6 - EXOC2 mRNA levels measured by qRT-PCT analysis of EXOC2 3933 with inhibited and uninhibited lysosomes. Relative EXOC2 mRNA levels were evaluated by qRT-PCT analysis for EXOC2 3933 cells with inhibited and uninhibited lysosomes (Figures 7 and 8).

[0268] Relative EXOC2 mRNA levels were analyzed by qRT-PCR in HEK cells treated with 5 μM NTC, EXOC2 targeting the gapmer ASO 3933, and LysoASO 3933-G5 for 2 days. HEK cells were treated with DMSO or 50 nM bafilomycin A1 (Baf A1) and lysosomal inhibitors for 24 hours prior to ASO treatment.

[0269] LysoASO was found to mediate its effects via functional lysosomes. Gapmer ASO 3933 was found to significantly reduce relative EXOC2 mRNA levels in DMSO and Baf A1-treated cells compared to NTC ASO. LysoASO 3933-G5 was found to further improve the knockdown efficiency of ASO 3933. EXOC2 mRNA levels were significantly reduced in LysoASO 3933-G5 compared to ASO 3933 in DMSO-treated cells. However, this effect was suppressed in Baf A1-treated cells, and LysoASO 3933-G5 did not further reduce EXOC2 mRNA levels compared to ASO 3933.

[0270] Example 7 - EXOC2 mRNA levels measured by qRT-PCT analysis of sterically blocked and unsterically blocked EXOC2 3933. The relative EXOC2 mRNA levels of sterically blocked and unsterically blocked EXOC2 3933 were evaluated by qRT-PCT analysis (Figures 9 and 10).

[0271] Relative EXOC2 mRNA levels were analyzed by qRT-PCR in HEK cells treated for 2 days with 5 μM NTC, EXOC2 targeting steric-blocking ASO 3933, and LysoASO 3933-G5 which steric-blocks ASO.

[0272] LysoASO was found to target an RNase H-independent pathway for RNA degradation. As expected, sterically, sterically barricaded ASO 3933 was found to not alter relative EXOC2 mRNA levels compared to NTC. However, sterically barricaded LysoASO 3933-G5 was found to significantly reduce EXOC2 mRNA levels compared to NTC, suggesting the involvement of an alternative degradation pathway to RNase H.

[0273] Example 8 - Ku80 mRNA levels measured by qRT-PCT analysis for Ku80 624 and 2802. The relative Ku80 mRNA levels of Ku80 624 and 2802 were evaluated by qRT-PCT analysis (Figure 11).

[0274] qRT-PCR analysis of relative Ku80 mRNA levels was performed on (A) iPSC-derived neurons treated with Ku80 targeting 1 μM NTC, gapmers ASO 624 and 1425, LysoASO 624-G10, and 1425-G10, and (B) HeLa cells treated for 2 days with Ku80 targeting 1 μM NTC, gapmers ASO 2802 and LysoASO 2802-G10.

[0275] LysoASO was found to effectively induce Ku80 mRNA for degradation. Gapmers ASO 624 and 1424 were found to significantly reduce Ku80 mRNA levels compared to NTC. LysoASO 624-G10 and 1425-G10 were found to further reduce Ku80 mRNA levels compared to gapmers ASO 624 and 1424, respectively. Gapmer ASO 2802 was found to not alter Ku80 mRNA levels compared to NTC. LysoASO 2802-G10 was found to significantly reduce Ku80 mRNA levels compared to NTC and ASO 2802.

[0276] Example 9 - TASK1 mRNA levels measured by qRT-PCT analysis for TASK1 622 and 5968. The relative TASK1 mRNA levels of TASK1 622 and 5968 were evaluated by qRT-PCT analysis (Figure 12).

[0277] qRT-PCR analysis of relative Task1 mRNA levels in iPSC-derived neurons treated with 5 μM NTC, targeting gapmers ASO 692 and 5968, as well as in LysoASO 692-G10 and 5968-G10, was performed over two days.

[0278] LysoASO was found to effectively induce Task1 mRNA for degradation. ASO 692 was found to not alter Task1 mRNA levels compared to NTC, while ASO 5968 was found to significantly reduce Task1 mRNA levels compared to NTC. LysoASO 692-G10 and 5968-G10 were found to further reduce Task1 mRNA levels compared to ASO 692 and 5968, respectively, and to enable the non-functional ASO 692.

[0279] Example 10 - Enhancement of silencing from approved oligonucleotide therapeutics with poly-G ligand The advantage of the poly-G ligand described herein is its broad utility for enhancing silencing in any therapeutic oligonucleotide, including approved oligonucleotide therapeutics already selected for their robust silencing effect. To demonstrate this utility, two approved oligonucleotide therapeutics, anti-SOD1 ASO tofersen (QALSODY®) and anti-PCSK9 siRNA inclisiran (LEQVIO®), were tested with and without the poly-G ligand.

[0280] Example 10 - Materials and Methods cell culture SH-SY5Y cells were maintained in DMEM / F-12 (Dulbeccoo's modified Eagle medium / nutrient mixture F-12) supplemented with 10% FBS (fetal bovine serum) and 1% penicillin / strepmycin (Pen-strep). A549 cells were maintained in F-12 medium supplemented with 10% FBS and 1% Pen-strep. Cells were divided into 2 × 10⁶ for treatment. 5 The cells were seeded in a 24-well plate at a density of one cell per well.

[0281] ASO treatment SH-SY5Y cells were transfected with 25 or 100 nM ASOs listed in Table 3 using Lipofectamine RNAiMAX. Cells were harvested 24 hours after ASO treatment for RNA extraction and RT-qPCR analysis.

[0282] siRNA treatment Sense siRNA was hybridized with either an antisense strand or a run-antisense strand. A549 cells were transfected with 50 nM siRNA listed in Table 4 using Lipofectamine RNAiMAX. Cells were harvested 48 hours after siRNA treatment for RNA extraction and RT-qPCR analysis.

[0283] RNA extraction and real-time quantitative PCR Total RNA was extracted using acid guanidinium thiocyanate phenol-chloroform extraction (QIAzol reagent, Qiagen) according to the manufacturer's protocol. 1 μg of RNA was reverse transcribed into cDNA using a random hexamer and ProtoScript II reverse transcriptase (New England Biolabs).

[0284] Real-time quantitative PCR (RT-qPCR) was performed using a Bio-rad CFX96 real-time PCR instrument. The Ct value of each sample was normalized to GAPDH. The relative mRNA levels of each gene were calculated as follows: -ΔΔCt The calculations were performed using the specified method. The qRT-PCR primers are listed in Table 5.

[0285] Example 10 - Results ASO tofersen was tested in SH-SY5Y cells treated with 25 or 100 nM of each compound, with and without poly-G ligand 10G (i.e., Lamp ligand). As shown in Figure 13, the silencing effect of tofersen was significantly improved by including the 10G ligand, based on relative SOD1 expression, compared to the untargeted control. An alternative poly-G ligand was also tested with tofersen (tofersen-Lamp derivative). This alternative poly-G ligand does not contain consecutive G nucleotides; rather, intervening non-G nucleotides (A nucleotides) are present every 3 G nucleotides. As shown in Figure 15, the alternative poly-G ligand had an effect of enhancing SOD1 suppression equivalent to that of the consecutive G ligand 10G.

[0286] siRNA inclisilane was also tested with and without poly-G ligand. In this case, 5G ligand was used at the 3' end of the antisense strand. A549 cells were treated with 50 nM inclisilane or inclisilane-Lamp for 48 hours. As shown in Figure 14, the silencing effect of inclisilane was also significantly improved by inclusion of poly-G ligand (5G ligand), based on relative PCSK9 expression compared to untargeted controls.

[0287] Importantly, both figures demonstrate the usefulness of poly-G ligands for enhancing silencing in two different therapeutic oligonucleotide types: ASOs and siRNAs. Furthermore, poly-G ligands enhanced silencing in therapeutic oligonucleotides containing a wide variety of chemical modifications.

[0288] This study demonstrates that poly-G ligands are broadly applicable to any oligonucleotide type, regardless of the underlying oligonucleotide sequence or chemical modification used.

[0289] array [Table 1-1] [Table 1-2] Note: (-) indicates a phosphodiester skeleton, and (*) indicates a phosphorothioate skeleton. Black (N) indicates an unmodified nucleotide, and bold italicized (N) indicates a nucleotide with 2'-O-methoxyethyl (2'MOE) sugar modification. [Table 2] [Table 3] [Table 4] [Table 5]

[0290] References Aboul-Fadl, T. (2018). Antisense Oligonucleotides: Strategies, Designs, and Principles. Methods in Molecular Biology, 1768, 3-23. Bennett, CF, & Swayze, EE (2010). RNA Targeting Therapeutics: Molecular Mechanisms of Antisense Oligonucleotides as a Therapeutic Platform. Annual Review of Pharmacology and Toxicology, 50, 259-293. Brummelkamp,T.R.,Bernards,R.,&Agami,R.(2002).A System for Stable Expression of Short Interfering RNAs in Mammalian Cells.Science,296(5567),550-553. Boudreau,R.L.,Spengler,R.M.,Hylock,R.H.,Kusenda,B.J.,Davis,H.A.,Eichmann,D.A.,...&Davidson,B.L.(2011).siSPOTR:A Tool for Designing Highly Specific and Potent siRNAs for Human and Mouse.Nucleic Acids Research,39(12),e82. Corey,D.R.(2017).Nusinersen,an Antisense Oligonucleotide Drug for Spinal Muscular Atrophy.Nature Neuroscience,20(4),497-499. Crooke,S.T.,Witztum,J.L.,Bennett,C.F.,&Baker,B.F.(2018).RNA-Targeted Therapeutics.Cell Metabolism,27(4),714-739. Daka,A.,Peer,D.,&Rothschild,G.(2020).Understanding Intracellular Barriers to mRNA-Based Therapeutics:A Panoramic Overview.Molecular Therapy,28(4),856-877. Elbashir,S.M.,Harborth,J.,Lendeckel,W.,Yalcin,A.,Weber,K.,&Tuschl,T.(2001).Duplexes of 21-Nucleotide RNAs Mediate RNA Interference in Cultured Mammalian Cells.Nature,411(6836),494-498. Fitzgerald,K.,Frank-Kamenetsky,M.,Shulga-Morskaya,S.,Liebow,A.,Bettencourt,B.R.,Sutherland,J.E.,...&Akinc,A.(2021).Effect of an RNA Interference Drug on the Synthesis of Proprotein Convertase Subtilisin / Kexin Type 9 (PCSK9) and the Concentration of Serum LDL Cholesterol in Healthy Volunteers:A Randomized,Single-Blind,Placebo-Controlled,Phase 1 Trial.The Lancet,383(9939),60-68. Fire,A.,Xu,S.,Montgomery,M.K.,Kostas,S.A.,Driver,S.E.,&Mello,C.C.(1998).Potent and Specific Genetic Interference by Double-Stranded RNA in Caenorhabditis Elegans.Nature,391(6669),806-811. Gilleron,J.,Querbes,W.,Zeigerer,A.,Borodovsky,A.,Marsico,G.,Schubert,U.,...&Akinc,A.(2013).Image-Based Analysis of Lipid Nanoparticle-Mediated siRNA Delivery,Intracellular Trafficking,and Endosomal Escape.Nature Biotechnology,31(7),638-646. Hannon,G.J.(2002).RNA Interference.Nature,418(6894),244-251. Jackson,A.L.,&Linsley,P.S.(2010).Recognizing and Avoiding siRNA Off-Target Effects for Target Identification and Therapeutic Application.Nature Reviews Drug Discovery,9(1),57-67. Juliano,R.L.(2016).The Delivery of Therapeutic Oligonucleotides.Nucleic Acids Research,44(14),6518-6548. Juliano,R.L.,&Ming,X.(2020).Carriers and Vehicles for Oligonucleotide Therapeutics.Molecular Therapy-Nucleic Acids,19,1338-1347. Khan,I.,Steeghs,K.,&Ngondo,R.P.(2021).Antisense Oligonucleotide Therapeutics:Focus on Gene Splicing.Journal of Controlled Release,329,476-495. Watts,J.K.,Deleavey,G.F.,Damha,M.J.,&Vasquez,G.(2018).Anthraquinone Conjugates of 2′-O-Methyl Oligonucleotides for Duplex and Triplex RNA Targeting.Nucleic Acids Research,46(11),5686-5696. Yuva-Aydemir,Y.,Almeida,S.,Krishnan,G.,Gendron,T.F.,and Gao,F-B.(2019).Transcription elongation factor AFF2 / FMR2 regulates expression of expanded GGGGCC repeat-containing C9ORF72 allele in ALS / FTD.Nat Commun.10,5466. Lopez-Gonzalez,R.,Yang,D.,Pribadi,M.,Kim,T.S.,Krishnan,G.,Choi,S.Y.,Lee,S.,Coppola,G.,and Gao,F-B.(2019).Partial inhibition of the overactivated Ku80-dependent DNA repair pathway rescues neurodegeneration in C9ORF72-ALS / FTD.PNAS 116,9628-9633. Capraro, V., Zane, L., Poncet, D., Perol, D., Galia, P., Preudhomme, C., Bonnefoy-Berard, N., Gilson, E., Thomas, X., El-Hamri, M., Chelghoun, Y., Michallet, M., Wattel, E., Mortreux, F., & Sibon, D. (2011). Telomere deregulations possess cytogenetic, phenotype, and prognostic specificities in acute leukemias. Experimental hematology, 39(2), 195-202.e2.

Claims

1. It is an oligonucleotide, The oligonucleotide comprises a 5' end, a 3' end, and complementarity to a target polynucleotide, wherein the oligonucleotide comprises a poly-G sequence ligated to the 5' end and / or the 3' end of the oligonucleotide, and the poly-G sequence lacks complementarity to the target polynucleotide.

2. The oligonucleotide according to claim 1, wherein the poly-G sequence comprises 2 to 30 G nucleotides.

3. The oligonucleotide according to claim 1 or 2, wherein the poly-G sequence is 2, 3, 4, 5, 6, 7, 8, 9, or 10 G nucleotides.

4. The oligonucleotide according to any one of claims 1 to 3, wherein the poly-G sequence comprises or consists of five G nucleotides.

5. The oligonucleotide according to any one of claims 1 to 3, wherein the poly-G sequence comprises or consists of six G nucleotides.

6. The oligonucleotide according to any one of claims 1 to 3, wherein the poly-G sequence comprises or consists of seven G nucleotides.

7. The oligonucleotide according to any one of claims 1 to 3, wherein the poly-G sequence comprises or consists of eight G nucleotides.

8. The oligonucleotide according to any one of claims 1 to 3, wherein the poly-G sequence comprises or consists of nine G nucleotides.

9. The oligonucleotide according to any one of claims 1 to 3, wherein the poly-G sequence comprises or consists of 10 G nucleotides.

10. The oligonucleotide according to any one of the prior claims, wherein the poly-G sequence comprises a G nucleotide that is continuous, discontinuous, or a combination thereof.

11. The oligonucleotide according to any one of claims 1 to 10, wherein the poly-G sequence includes or consists of (dG)(dG)(dG)(dG)(dG).

12. The oligonucleotide according to any one of claims 1 to 10, wherein the poly-G sequence includes or consists of (dG)(dG)(dG)(dG)(dG)(dG).

13. The oligonucleotide according to any one of claims 1 to 10, wherein the poly-G sequence includes or consists of (dG)(dG)(dG)(dG)(dG)(dG).

14. The oligonucleotide according to any one of claims 1 to 10, wherein the poly-G sequence comprises or consists of (dG)(dG)(dG)(dG)(dG)(dG)(dG).

15. The oligonucleotide according to any one of claims 1 to 10, wherein the polyG sequence comprises or consists of (dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG).

16. The oligonucleotide according to any one of claims 1 to 10, wherein the polyG sequence comprises or consists of (dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG).

17. The oligonucleotide according to any one of claims 1 to 10, wherein the polyG sequence comprises or consists of (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dN)(dG), each dN corresponding individually to a deoxyribonucleotide of dA, dT, or dC.

18. The oligonucleotide according to any one of claims 1 to 10, wherein the polyG sequence comprises or consists of (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dN)(dG)(dG), each dN corresponding individually to a deoxyribonucleotide of dA, dT, or dC.

19. The oligonucleotide according to any one of claims 1 to 10, wherein the polyG sequence comprises or consists of (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dN)(dG)(dG), each dN corresponding individually to a deoxyribonucleotide of dA, dT, or dC.

20. The oligonucleotide according to any one of the prior claims, wherein the oligonucleotide has a length of about 10 to about 35 nucleotides.

21. The oligonucleotide according to any one of the prior claims, wherein the oligonucleotide has a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

22. The oligonucleotide and / or the poly-G sequence comprises one or more modified nucleotides, as described in any one of the prior claims.

23. The oligonucleotide according to claim 22, wherein each of the one or more modified nucleotides independently comprises a modification of a ribose group, a phosphate group, a nucleic acid base, or a combination thereof.

24. The oligonucleotide according to claim 23, wherein each modification of the ribose group includes 2'-O-methyl, 2'-fluoro, 2'-deoxy, 2'-O-(2-methoxyethyl) (MOE), 2'-O-alkyl, 2'-O-alkoxy, 2'-O-alkylamino, 2'-NH2, a restricted nucleotide, or a combination thereof.

25. The oligonucleotide according to claim 24, wherein the restricted nucleotide includes Loc nucleic acid (LNA), ethyl restricted nucleotide, 2'-(S)-restricted ethyl (S-cEt) nucleotide, restricted MOE, 2'-O,4'-C-aminomethylene crosslinked nucleic acid (2',4'-BNANC), alpha-L-Loc nucleic acid, tricyclo-DNA, or a combination thereof.

26. The oligonucleotide according to claim 24, wherein the modification of the ribose group includes a 2'-O-(2-methoxyethyl) (MOE) modification.

27. The oligonucleotide according to claim 24, wherein the nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and / or 10 from the 5' end and / or 3' end of the oligonucleotide are 2'-O-(2-methoxyethyl) (MOE) modified.

28. The oligonucleotide according to claim 24, wherein all nucleotides of the oligonucleotide and / or the poly-G sequence include a 2'-O-(2-methoxyethyl) (MOE) modification.

29. The oligonucleotide according to claim 23, wherein the modification of the ribose group includes tricyclo-DNA modification.

30. The oligonucleotide according to claim 23, wherein all nucleotides of the oligonucleotide and / or the poly-G sequence include tricyclo-DNA modifications.

31. The oligonucleotide according to claim 23, wherein the modification of the ribose group includes a 2'-deoxy modification.

32. The oligonucleotide according to claim 23, wherein each modification of the phosphate group includes a phosphorothioate, phosphonoacetic acid (PACE), thiophosphonoacetic acid (thioPACE), amide, triazole, phosphonate, phosphotryester, or a combination thereof.

33. The oligonucleotide according to claim 23, wherein the modification of the phosphate group is a phosphorothioate.

34. The oligonucleotide according to claim 23, wherein all nucleotides of the oligonucleotide and / or the poly-G sequence contain a phosphorothioate.

35. The oligonucleotide according to any one of the prior claims, wherein the oligonucleotide and / or the poly-G sequence comprises at least one phosphodiester nucleotide linkage.

36. The oligonucleotide according to any one of the prior claims, wherein all internucleotide links in the oligonucleotide and / or the poly-G sequence are phosphodiester nucleotide links.

37. The oligonucleotide according to claim 23, wherein each modification of the nucleic acid base includes 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, halogenated aromatic group, or a combination thereof.

38. The oligonucleotide according to claim 23, wherein the modification of the nucleic acid base group includes a 5-methylcytosine modification.

39. An oligonucleotide according to any one of the prior claims, comprising a mixture of modified nucleotides.

40. The oligonucleotide according to any one of the prior claims, wherein the functionalized portion is linked to the 5' end or the 3' end of the oligonucleotide.

41. The oligonucleotide according to claim 40, wherein the functionalized portion comprises an N-acetylgalactosamine (GalNAc) portion and / or a hydrophobic portion.

42. The oligonucleotide according to claim 40, wherein the hydrophobic portion is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and mixtures thereof, optionally the steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA), and optionally the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanic acid (DCA).

43. formula: Including A-B-C, in the formula, A contains approximately 0 to 8 modified nucleotides. B contains approximately 6 to 18 deoxyribonucleic acid (DNA) nucleotides and / or DNA-like nucleotides. C contains approximately 0 to 8 modified nucleotides. The oligonucleotide according to any one of the prior claims, wherein the total length of the antisense oligonucleotide is approximately 10 to approximately 30 nucleotides.

44. The oligonucleotide according to claim 43, wherein A comprises about 2 to about 6 modified nucleotides, B comprises about 6 to about 12 DNA nucleotides and / or DNA-like nucleotides, and C comprises about 2 to about 6 modified nucleotides.

45. The oligonucleotide according to claim 43 or 44, wherein A comprises about five modified nucleotides, B comprises about ten DNA nucleotides and / or DNA-like nucleotides, and C comprises about five modified nucleotides.

46. The oligonucleotide according to claim 43 or 44, wherein A comprises about 2 to about 6 2'-O-(2-methoxyethyl) (MOE) modified nucleotides, B comprises about 6 to about 12 DNA-like nucleotides, and C comprises about 2 to about 6 2'-O-(2-methoxyethyl) (MOE) modified nucleotides.

47. The oligonucleotide according to claim 43 or 44, wherein A comprises about five 2'-O-(2-methoxyethyl) (MOE) modified nucleotides, B comprises about ten DNA-like nucleotides, and C comprises about five 2'-O-(2-methoxyethyl) (MOE) modified nucleotides.

48. The oligonucleotide according to any one of the prior claims, comprising a nucleic acid sequence having at least 90% sequence identity with any one of the nucleic acid sequences in Table 1.

49. XXSXXSXXSXXSXX Ｓ X Ｓ X Ｓ X Ｓ X Ｓ X Ｓ X Ｓ X Ｓ X Ｓ X Ｓ X Ｓ including an array modification pattern of XXSXXSXXSXXSXX, wherein s represents the linkage between phosphorothioate nucleosides, X contains adenosine, guanosine, cytidine, thymine, or uracil, and X contains a 2'-O-(2-methoxyethyl) modification. X comprises adenosine, guanosine, cytidine, thymine, or uracil, and X comprises a 2'-deoxy modification, as described in any one of the prior claims.

50. X S X S X S X S X Ｓ The formula includes the array modification pattern X S X S X S X S X S X S X S X S X S X S X S X S X, s represents the linkage between phosphorothioate nucleosides, The oligonucleotide according to any one of the prior claims, wherein X comprises adenosine, guanosine, cytidine, thymine, or uracil, and X comprises a 2'-O-(2-methoxyethyl) modification.

51. The oligonucleotide according to any one of the prior claims, wherein the target is mammalian or viral mRNA, and optionally, the target is an intron region or an exon region of the mRNA.

52. The target is an oligonucleotide according to any one of the prior claims, selected from the group consisting of the EXOC2 gene, the Ku80 gene, and the Task1 gene.

53. The oligonucleotide according to claim 52, wherein the oligonucleotide inhibits the expression of the EXOC2 gene, the Ku80 gene, or the Task1 gene by at least about 50%.

54. The oligonucleotide according to any one of claims 1 to 51, wherein the target is SOD1.

55. An oligonucleotide according to any one of claims 1 to 51, comprising the sequence (eC)#(eA)(eG)#(eG)(eA)#(dT)#(dA)#(d5C)#(dA)#(dT)#(dT)#(dT)#(d5C)#(dT)#(dA)#(eC)(eA)#(eG)(eC)#(eU)(polyG), wherein (#) indicates a phosphorothioate linkage, (e) indicates a 2'MOE modification, (d) indicates a deoxyribonucleotide, (dN) indicates a deoxyribonucleotide of A, T, or C, (d5C) indicates 5-methylcytosine, and (polyG) indicates 2 to 30 G nucleotides.

56. Sequence (eC)#(eA)(eG)#(eG)(eA)#(dT)#(dA)#(d5C)#(dA)#(dT)#(dT)#(dT)#(d5C)#(dT)#(dA)#(eC)(eA)#(eG)(eC)#(eU)(dG) ｘ An oligonucleotide according to any one of claims 1 to 51, comprising, in the formula, (#) represents a phosphorothioate linkage, (e) represents a 2'MOE modification, (d) represents a deoxyribonucleotide, (d5C) represents 5-methylcytosine, and x represents an integer from 5 to 10.

57. Sequence (eC) #(eA) (eG) #(eG) (eA) #(dT) #(dA) #(d5C) #(dA) #(dT) #(dT) #(dT) #(d5C) #(dT) #(dA) #(eC)(eA) #(eG)(eC) #(eU)(dGdGdGdN) ｘ The oligonucleotide according to any one of claims 1 to 51, comprising, in the formula, (#) represents a phosphorothioate linkage, (e) represents a 2'MOE modification, (d) represents a deoxyribonucleotide, (d5C) represents 5-methylcytosine, (dN) represents a deoxyribonucleotide of A, T, or C, and x represents an integer from 2 to 10.

58. The oligonucleotide according to any one of claims 1 to 51, wherein the target is PCSK9.

59. The oligonucleotide according to any one of claims 1 to 51, wherein the oligonucleotide is selected from the group consisting of antisense oligonucleotides (ASO), gapmers, siRNA, miRNA, shRNA, CRISPR guide, DNA, antisense mixmers, miRNA inhibitors, splice-switching oligonucleotides (SSO), phosphorodiamidate morpholino oligomers (PMO), and peptide nucleic acids (PNA).

60. The oligonucleotide according to any one of claims 1 to 42, wherein the oligonucleotide is double-stranded RNA (dsRNA).

61. An antisense chain complementary to the target, wherein the antisense chain is optionally a chain having a length of approximately 10 to 35 nucleotides, and A sense strand that is complementary to at least a portion of the antisense strand, and optionally the sense strand having a length of about 10 to 35 nucleotides, The dsRNA according to claim 60, comprising a poly-G sequence ligated to the 5' and / or 3' ends of the antisense strand and / or the sense strand.

62. The dsRNA according to claim 60 or 61, wherein the target is PCSK9.

63. V(mA)#(fC)#(mA)(fA)(fA)(fA)(mG)(fC)(mA)(fA)(mA)(mC)(fA)(mG)(fG)(mU)(fC)(mU)(mA)(mG)#(mA)#(mA) and (mC)#(mU)#(mA)(mG)(mA)(mC)(fC)(mU)(fG)(mU)(dT)(mU)(mU)(mG)(mC)(mU) dsRNA according to any one of claims 60 to 62, comprising a sense strand of (mU)(mU)(mU)(mG)(mU)GalNac, wherein (#) indicates phosphorothioate linkage, (mN) indicates 2'-OMe modification, (fN) indicates 2'-fluoro modification, V indicates 5'-vinyl phosphate, and GalNac indicates an N-acetylgalactosamine (GalNAc) conjugate.

64. dsRNA according to any one of claims 60 to 63, comprising an antisense strand of V(mA)#(fC)#(mA)(fA)(fA)(fA)(mG)(fC)(mA)(fA)(mA)(mC)(fA)(mG)(fG)(mU)(fC)(mU)(mA)(mG)#(mA)#(mA)(polyG), wherein (#) indicates phosphorothioate linkage, (mN) indicates 2'-OMe modification, (fN) indicates 2'-fluoro modification, V indicates 5'-vinyl phosphate, and (polyG) indicates 2 to 30 G nucleotides.

65. V (mA) # (fC) # (mA) (fA) (fA) (fA) (mG) (fC) (mA) (fA) (mA) (mA) (mC) (fA) (mG) (fG) (mU) (fC) (mU) (mA) (mG) # (mA) # (mA) (dG) ｘ dsRNA according to any one of claims 60 to 63, comprising an antisense strand, wherein (#) represents phosphorothioate linkage, (mN) represents 2'-OMe modification, (fN) represents 2'-fluoro modification, V represents 5'-vinyl phosphate, and x represents an integer from 5 to 10.

66. A pharmaceutical composition for inhibiting gene expression in living organisms, The pharmaceutical composition comprising an oligonucleotide according to any one of claims 1 to 59 or a dsRNA according to any one of claims 60 to 65, and a pharmaceutically acceptable carrier.

67. The pharmaceutical composition according to claim 66, wherein the gene is selected from the group consisting of the EXOC2 gene, the Ku80 gene, and the Task1 gene.

68. The pharmaceutical composition according to claim 67, wherein the oligonucleotide or the dsRNA inhibits the expression of the EXOC2 gene, the Ku80 gene, or the Task1 gene by at least about 50%.

69. The pharmaceutical composition according to claim 68, wherein the oligonucleotide or the dsRNA inhibits the expression of the EXOC2 gene, the Ku80 gene, or the Task1 gene by at least about 80%.

70. A vector comprising a regulatory sequence operably ligated to a nucleotide sequence encoding an oligonucleotide according to any one of claims 1 to 59 or a dsRNA according to any one of claims 60 to 65.

71. The vector according to claim 70, wherein the oligonucleotide or the dsRNA inhibits gene expression by at least 30%.

72. The vector according to claim 70, wherein the oligonucleotide or the dsRNA inhibits gene expression by at least about 50%.

73. The vector according to claim 70, wherein the oligonucleotide or the dsRNA inhibits gene expression by at least about 80%.

74. The vector according to any one of claims 70 to 73, wherein the gene is selected from the group consisting of the EXOC2 gene, the Ku80 gene, and the Task1 gene.

75. Recombinant adeno-associated virus (rAAV), comprising the vector and AAV capsid described in any one of claims 70 to 74.

76. A cell comprising the vector according to any one of claims 70 to 74 or the rAAV according to claim 75.

77. A method for inhibiting gene expression in cells, wherein the method is (a) The step of introducing an oligonucleotide according to any one of claims 1 to 59, a dsRNA according to any one of claims 60 to 65, a vector according to any one of claims 70 to 74, or an rAAV according to claim 75 into the cells, (b) The method comprising the step of maintaining the cells generated in step (a) for a time sufficient to achieve degradation of the mRNA transcript of the gene, thereby inhibiting the expression of the gene in the cells.

78. The method according to claim 77, wherein the poly-G sequence induces degradation from lysosomes.

79. The method according to claim 78, wherein the gene is selected from the group consisting of the EXOC2 gene, the Ku80 gene, and the Task1 gene.

80. The method according to claim 79, wherein the oligonucleotide, the dsRNA, the vector, or the rAAV inhibits the expression of the EXOC2 gene, the Ku80 gene, or the Task1 gene by at least about 50%.

81. The method according to claim 80, wherein the oligonucleotide, the dsRNA, the vector, or the rAAV inhibits the expression of the EXOC2 gene, the Ku80 gene, or the Task1 gene by at least about 80%.

82. The method according to any one of claims 77 to 81, wherein the oligonucleotide, the dsRNA, the vector, or the rAAV is administered by intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof.

83. A method for treating or managing a gene-related disorder, The method comprising the step of administering a therapeutically effective amount of an oligonucleotide according to any one of claims 1 to 59, a dsRNA according to any one of claims 60 to 65, a vector according to any one of claims 70 to 74, or an rAAV according to claim 75 to a patient in need of such treatment.

84. The method according to claim 83, wherein the gene is selected from the group consisting of the EXOC2 gene, the Ku80 gene, and the Task1 gene.

85. The method according to claim 84, wherein the oligonucleotide, the dsRNA, the vector, or the rAAV inhibits the expression of the EXOC2 gene, the Ku80 gene, or the Task1 gene by at least about 50%.

86. The method according to claim 85, wherein the oligonucleotide, the dsRNA, the vector, or the rAAV inhibits the expression of the EXOC2 gene, the Ku80 gene, or the Task1 gene by at least about 80%.

87. A method for degrading a target polynucleotide in the lysosome of a cell, wherein the method is The method comprising the steps of introducing an oligonucleotide according to any one of claims 1 to 59, a dsRNA according to any one of claims 60 to 65, a vector according to any one of claims 70 to 74, or an rAAV according to claim 75 into the cells, and maintaining the cells for a time sufficient to degrade the target polynucleotide in the lysosomes of the cells.

88. The method according to claim 87, wherein the target is mammalian or viral mRNA, and optionally, the target is an intron region or an exon region of the mRNA.

89. The method according to claim 88, wherein the target is selected from the group consisting of the EXOC2 gene, the Ku80 gene, and the Task1 gene.

90. The method according to claim 89, wherein the oligonucleotide inhibits the expression of the EXOC2 gene, the Ku80 gene, or the Task1 gene by at least about 50%.

91. A method for treating or managing amyotrophic lateral sclerosis (ALS) in patients, The method comprising the step of administering to the patient a therapeutically effective amount of the oligonucleotide according to any one of claims 54 to 57.

92. A method for treating or managing primary hyperlipidemia in patients, The method comprising the step of administering to the patient a therapeutically effective amount of the dsRNA according to any one of claims 62 to 65.

93. The method according to claim 92, wherein the primary hyperlipidemia is heterozygous familial hypercholesterolemia (HeFH).

94. A method for lowering a patient's low-density lipoprotein cholesterol (LDL-C), The method comprising the step of administering to the patient a therapeutically effective amount of the dsRNA described in any one of claims 62 to 65.

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