Degradation of rna by the lysosomal pathway

Abstract

The present disclosure provides oligonucleotides, methods, and compositions for degrading RNA via the lysosomal pathway. Also encompassed are oligonucleotides, methods, and compositions for treating, preventing, or ameliorating diseases, disorders, and conditions associated with EXOC2 , Ku80 and Task1 in a subject in need thereof.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 532,208, filed August 11, 2023. The entire contents of the above-mentioned referenced patent application are incorporated herein by reference in their entirety. Background Technology

[0003] RNA knockdown-based methods have become powerful tools for manipulating gene expression and elucidating gene function (Fire et al., 1998; Hannon, 2002). These methods utilize small RNA molecules, such as small interfering RNA (siRNA) and short hairpin RNA (shRNA), offering the possibility of silencing gene expression with high precision and specificity (Elbashir et al., 2001; Brummelkamp et al., 2002). Despite their promising prospects, these methods still face challenges, particularly in terms of efficacy and target specificity (Jackson & Linsley, 2010; Boudreau et al., 2011).

[0004] One of the key limitations encountered in RNA knockdown methods relates to the intracellular fate of RNA molecules. Cellular uptake and subsequent processing are complex processes that can profoundly affect knockdown efficiency (Juliano, 2016). Furthermore, poor endosome escape and premature degradation in the lysosomal compartment have been identified as major obstacles to the effectiveness of these methods (Gilleron et al., 2013; Daka et al., 2020).

[0005] Therefore, more efficient RNA knockdown methods are needed. Summary of the Invention

[0006] This disclosure provides oligonucleotides, methods, and compositions for degrading RNA via the lysosomal pathway.

[0007] In one aspect, this disclosure provides an oligonucleotide comprising a 5' end, a 3' end, and complementarity to a target polynucleotide, wherein the oligonucleotide comprises a polyG sequence linked to the 5' end and / or the 3' end of the oligonucleotide, and wherein the polyG sequence lacks complementarity to the target polynucleotide.

[0008] In some implementations, the poly-G sequence contains 2-30 G nucleotides.

[0009] In some embodiments, the poly-G sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 5 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 6 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 7 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 8 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 9 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 10 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 11 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 12 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 13 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 14 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 15 G nucleotides.

[0010] In some embodiments, the polyG sequence is linked to the 3' end of the oligonucleotide of this disclosure. In other embodiments, the polyG sequence is linked to the 5' end of the oligonucleotide of this disclosure. In other embodiments, the polyG sequence is linked to both the 5' and 3' ends of the oligonucleotide of this disclosure.

[0011] In some implementations, the polyG sequence is single-stranded.

[0012] In some embodiments, the polyG sequence binds to proteins on the surface of the lysosome. In some embodiments, the polyG sequence binds to a lysosome-associated membrane glycoprotein (LAMP). In some embodiments, the LAMP is LAMP2C.

[0013] In some embodiments, the poly-G sequence comprises consecutive G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 5-15 consecutive G nucleotides. In some embodiments, the poly-G sequence comprises one or more non-G nucleotides (e.g., A, T, U, or C) within the poly-G sequence. In some embodiments, the oligonucleotide comprises two or more poly-G sequences, with one or more non-G nucleotides (e.g., A, T, U, or C) between the two or more poly-G sequences.

[0014] In some implementations, the polyG sequence comprises continuous, discontinuous, or combinations thereof G nucleotides.

[0015] In some embodiments, the polyG sequence comprises or consists of (dG)(dG)(dG)(dG)(dG)(dG). (This text is repeated 6 times in the original.) In some embodiments, the polyG sequence comprises or is composed of (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dG)(dN)(dG), wherein each dN individually corresponds to a deoxyribonucleotide of dA, dT, or dC. In some embodiments, the polyG sequence comprises or is composed of (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dG)(dN)(dG)(dG), wherein each dN individually corresponds to a deoxyribonucleotide of dA, dT, or dC. In some embodiments, the polyG sequence comprises or is composed of (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dG)(dN)(dG)(dG), wherein each dN individually corresponds to a deoxyribonucleotide of dA, dT, or dC.

[0016] In some implementations, the oligonucleotide is about 10 nucleotides to about 35 nucleotides in length.

[0017] In some implementations, the oligonucleotide is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 nucleotides in length.

[0018] In some implementations, the oligonucleotide and / or polyG sequence contains one or more modified nucleotides.

[0019] In some embodiments, one or more modified nucleotides each independently contain modifications of a ribose group, a phosphate group, a nucleobase, or a combination thereof.

[0020] In some embodiments, each modification of the ribosome group comprises 2'-O-methyl, 2'-fluorine, 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.

[0021] In some embodiments, the binding nucleotide comprises locked nucleic acid (LNA), ethyl-bound nucleotide, 2'-(S)-bound ethyl (S-cEt) nucleotide, bound MOE, 2'-O,4'-C-aminomethylene bridging nucleic acid (2',4'-BNANC), α-L-locked nucleic acid, tricyclic DNA, or combinations thereof.

[0022] In some embodiments, the modification of the ribose group includes 2'-O-(2-methoxyethyl) (MOE) modification.

[0023] In some embodiments, 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 contain 2'-O-(2-methoxyethyl) (MOE) modification.

[0024] In some embodiments, each nucleotide of the oligonucleotide and / or polyG sequence contains a 2'-O-(2-methoxyethyl) (MOE) modification.

[0025] In some implementations, the modification of the ribosome group includes tricyclic DNA modification.

[0026] In some implementations, each nucleotide of the oligonucleotide and / or polyG sequence contains a tricyclic DNA modification.

[0027] In some implementations, the modification of the ribosome group includes 2'-deoxy modification.

[0028] In some embodiments, each modification of the phosphate group comprises a thiophosphate, a phosphonoacetate (PACE), a thiophosphonoacetate (thioPACE), an amide, a triazole, a phosphonate, a triphosphate, or a combination thereof.

[0029] In some implementations, the phosphate group is modified to a thiophosphate ester.

[0030] In some implementations, each nucleotide of the oligonucleotide and / or polyG sequence contains a phosphate thioester.

[0031] In some implementations, the oligonucleotide and / or polyG sequence contains at least one phosphodiester nucleotide inter-bond.

[0032] In some implementations, each nucleotide bond in the oligonucleotide and / or polyG sequence is a phosphodiester nucleotide bond.

[0033] In some embodiments, each modification of the nucleobase comprises 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.

[0034] In some embodiments, the modification of the nucleobase group includes 5-methylcytosine modification.

[0035] In some implementations, the oligonucleotide comprises a mixture of modified nucleotides.

[0036] In some implementations, the functional portion is linked to the 5' or 3' end of the oligonucleotide.

[0037] In some implementations, the functional portion includes an N-acetylgalactosamine (GalNAc) portion and / or a hydrophobic portion.

[0038] In some implementations, the hydrophobic portion is selected from the group consisting of: fatty acids, steroids, open-ring steroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and mixtures thereof.

[0039] In some implementations, the steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA).

[0040] In some implementations, the fatty acids are selected from the group consisting of: eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanoic acid (DCA).

[0041] In some implementations, the oligonucleotide comprises the following formula: ABC, where: A contains approximately 0 to approximately 8 modified nucleotides; B contains approximately 6 to approximately 18 deoxyribonucleic acid (DNA) nucleotides and / or DNA-like nucleotides; and C contains approximately 0 to approximately 8 modified nucleotides; Furthermore, the full length of the antisense oligonucleotides mentioned therein is approximately 10 to approximately 30 nucleotides.

[0042] 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.

[0043] In some implementations, A contains about 5 modified nucleotides, B contains about 10 DNA nucleotides and / or DNA-like nucleotides, and C contains about 5 modified nucleotides.

[0044] In some embodiments, A comprises about 2 to about 6 nucleotides modified with 2'-O-(2-methoxyethyl) (MOE), B comprises about 6 to about 12 DNA-like nucleotides, and C comprises about 2 to about 6 nucleotides modified with 2'-O-(2-methoxyethyl) (MOE).

[0045] In some embodiments, A contains about 5 2'-O-(2-methoxyethyl) (MOE) modified nucleotides, B contains about 10 DNA-like nucleotides, and C contains about 5 2'-O-(2-methoxyethyl) (MOE) modified nucleotides.

[0046] In some implementations, the oligonucleotide comprises a nucleic acid sequence that has at least 90% sequence identity with any of the nucleic acid sequences in Table 1.

[0047] In some implementations, the oligonucleotide contains 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 X S X The sequence modification pattern, where s represents the internucleotide bond between thiophosphates; X It contains adenosine, guanosine, cytidine, thymine, or uracil, among which X Contains 2'- O -(2-methoxyethyl) modified; and X contains adenosine, guanosine, cytidine, thymine, or uracil, wherein X contains a 2'-deoxy modification.

[0048] In some implementations, the oligonucleotide contains 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 sequence modification pattern, where 's' represents the nucleotide internucleotide bond of a thiophosphate ester; and X It contains adenosine, guanosine, cytidine, thymine, or uracil, among which X Contains 2'- O -(2-methoxyethyl) modification.

[0049] In some implementations, the oligonucleotide contains 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 X S X The sequence modification pattern, where 's' represents the nucleotide internucleotide bond of a thiophosphate ester; and X It contains adenosine, guanosine, cytidine, thymine, or uracil, among which X Contains 2'- O -(2-methoxyethyl) modification.

[0050] In some embodiments, the target polynucleotide is mammalian or viral mRNA. In some embodiments, the target polynucleotide is an intron or exon region of mRNA.

[0051] In some implementations, the target is selected from the following group: EXOC2 Gene, Ku80 Genes and Task 1 Gene.

[0052] In some implementations, oligonucleotides will EXOC2 Gene, Ku80 Gene or Task 1 Gene expression is suppressed by at least approximately 50%.

[0053] In some implementation schemes, the target is SOD1 .

[0054] In some embodiments, the oligonucleotide comprises the sequence (eC)#(eA)(eG)#(eG)(eA)#(dT)#(dA)#(d5C)#(dA)#(dT)#(dT)#(d5C)#(dT)#(dA)#(eC)(eA)#(eG)(eC)#(eU)(polyG), wherein (#) indicates a phosphate thioester bond, (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 2-30 G nucleotides.

[0055] In some implementations, the oligonucleotide comprises the sequence (eC)#(eA)(eG)#(eG)(eA)#(dT)#(dA)#(d5C)#(dA)#(dT)#(dT)#(d5C)#(dT)#(dA)#(eC)(eA)#(eG)(eC)#(eU)(dG x , where (#) indicates a thiophosphate bond, (e) indicates a 2'MOE modification, (d) indicates a deoxyribonucleotide, (d5C) indicates 5-methylcytosine, and x indicates an integer between 5 and 10.

[0056] In some implementations, the oligonucleotide comprises the sequence (eC)#(eA)(eG)#(eG)(eA)#(dT)#(dA)#(d5C)#(dA)#(dT)#(dT)#(d5C)#(dT)#(dA)#(eC)(eA)#(eG)(eC)#(eU)(dGdGdGdN) x , where (#) indicates a thiophosphate bond, (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 between 2 and 10.

[0057] In some implementation schemes, the target is PCSK9 .

[0058] In some implementations, the oligonucleotide is selected from the group consisting of: antisense oligonucleotides (ASO), gapmers, siRNA, miRNA, shRNA, CRISPR guides, DNA, antisense hybrids, miRNA inhibitors, splice-conversion oligonucleotides (SSO), diaminophosphate morpholino oligomers (PMO), and peptide nucleic acids (PNA).

[0059] In some implementations, the oligonucleotide is double-stranded RNA (dsRNA).

[0060] In some embodiments, the dsRNA comprises an antisense strand complementary to the target polynucleotide. In some embodiments, the antisense strand is about 10-35 nucleotides in length. In some embodiments, the antisense strand is 18, 19, 20, 21, 22, or 23 nucleotides in length. In some embodiments, the dsRNA comprises a sense strand complementary to at least a portion of the antisense strand. In some embodiments, the sense strand is about 10-35 nucleotides in length. In some embodiments, the sense strand is 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.

[0061] In some implementations, the polyG sequence is attached to the 5' and / or 3' ends of the antisense and / or sense strands.

[0062] In some implementation schemes, the target is PCSK9 .

[0063] In some embodiments, the dsRNA comprises an antisense strand of V(mA)#(fC)#(mA)(fA)(fA)(fA)(mG)(fC)(mA)(fA)(mA)(mA)(mC)(fA)(mG)(fG)(mU)(fC)(mU)(mA)(mG)#(mA)#(mA) and a sense strand of (mC)#(mU)#(mA)(mG)(mA)(mC)(fC)(mU)(fG)(mU)(dT)(mU)(mU)(mG)(mC)(mU)(mU)(mU)(mU)(mG)(mU)(mG)(mU)GalNac, wherein (#) indicates a thiophosphate bond, (mN) indicates 2'-OMe modification, (fN) indicates 2'-fluorine modification, V indicates 5'-vinyl phosphate, and GalNAc indicates an N-acetylgalactosamine (GalNAc) conjugate.

[0064] In some implementations, the dsRNA contains an antisense strand of V(mA)#(fC)#(mA)(fA)(fA)(fA)(mG)(fC)(mA)(fA)(mA)(mA)(mC)(fA)(mG)(fG)(mU)(fC)(mU)(mA)(mG)#(mA)#(mA)(polyG), where (#) indicates a thiophosphate bond, (mN) indicates 2'-OMe modification, (fN) indicates 2'-fluorine modification, V indicates 5'-vinyl phosphate, and (polyG) indicates 2-30 G nucleotides.

[0065] In some implementations, the dsRNA contains of V(mA)#(fC)#(mA)(fA)(fA)(fA)(mG)(fC)(mA)(fA)(mA)(mA)(mA)(mC)(fA)(mG)(fG)(mU)(fC)(mU)(mA)(mG)#(mA)#(mA)(dG) x The antisense chain, where (#) indicates a thiophosphate bond, (mN) indicates 2'-OMe modification, (fN) indicates 2'-fluorine modification, V indicates 5'-vinylphosphine, and x indicates an integer between 5 and 10.

[0066] In another aspect, this disclosure provides a pharmaceutical composition for inhibiting gene expression in an organism, the pharmaceutical composition comprising oligonucleotides or dsRNA and a pharmaceutically acceptable carrier.

[0067] In some implementations, the genes are selected from the following groups: EXOC2 Gene, Ku80 Genes and Task 1 Gene.

[0068] In some implementations, oligonucleotides or dsRNA will EXOC2Gene, Ku80 Gene or Task 1 Gene expression is suppressed by at least approximately 50%.

[0069] In some implementations, oligonucleotides or dsRNA will EXOC2 Gene, Ku80 Gene or Task 1 Gene expression is suppressed by at least approximately 80%.

[0070] In another aspect, this disclosure provides a vector comprising a regulatory sequence operatively linked to a nucleotide sequence encoding an oligonucleotide or dsRNA.

[0071] In some implementations, oligonucleotides or dsRNAs suppress gene expression by at least 30%.

[0072] In some implementations, oligonucleotides or dsRNAs suppress gene expression by at least about 50%.

[0073] In some implementations, oligonucleotides or dsRNAs suppress gene expression by at least about 80%.

[0074] In some implementations, the genes are selected from the following groups: EXOC2 Gene, Ku80 Genes and Task 1 Gene.

[0075] In another aspect, this disclosure provides a recombinant adeno-associated virus (rAAV) comprising a vector and an AAV capsid.

[0076] In another aspect, this disclosure provides a cell comprising a vector or rAAV.

[0077] In another aspect, this disclosure provides a method for suppressing gene expression in cells, the method comprising: (a) Introducing oligonucleotides, dsRNA, vectors, or rAAV into cells; and (b) The cells produced in step (a) are maintained for a time sufficient to allow the mRNA transcript of the gene to degrade, thereby inhibiting the expression of the gene in the cells.

[0078] In some implementations, the polyG sequence guides degradation from lysosomes.

[0079] In some implementations, the genes are selected from the following groups: EXOC2 Gene, Ku80 Genes and Task 1 Gene.

[0080] In some implementations, oligonucleotides, dsRNA, vectors, or rAAV will EXOC2 Gene, Ku80 Gene or Task 1 Gene expression is suppressed by at least approximately 50%.

[0081] In some implementations, oligonucleotides, dsRNA, vectors, or rAAV will EXOC2 Gene, Ku80 Gene or Task 1 Gene expression is suppressed by at least approximately 80%.

[0082] In some implementations, oligonucleotides, dsRNA, vectors, or rAAV are administered via intravenous (IV), subcutaneous (SQ), or combinations thereof.

[0083] In another aspect, this disclosure provides a method for treating or managing a gene-related disease, the method comprising administering a therapeutically effective amount of an oligonucleotide, dsRNA, vector, or rAAV to a patient in need of such treatment.

[0084] In some implementations, the genes are selected from the following groups: EXOC2 Gene, Ku80 Genes and Task 1 Gene.

[0085] In some implementations, oligonucleotides, dsRNA, vectors, or rAAV will EXOC2 Gene, Ku80 Gene or Task 1 Gene expression is suppressed by at least approximately 50%.

[0086] In some implementations, oligonucleotides, dsRNA, vectors, or rAAV will EXOC2 Gene, Ku80 Gene or Task 1 Gene expression is suppressed by at least approximately 80%.

[0087] In another aspect, this disclosure provides a method for degrading a target polynucleotide in the lysosomes of a cell, the method comprising introducing the oligonucleotide described herein, the dsRNA described herein, the vector described herein, or the rAAV described herein into a cell, and maintaining the cell for a time sufficient for the target polynucleotide to be degraded in the lysosomes of the cell.

[0088] In some implementations, the method for degrading the target polynucleotide in the lysosomes of the cell occurs in vivo, in vitro, or outside the body.

[0089] In some embodiments, the target polynucleotide is mammalian or viral mRNA. In some embodiments, the target polynucleotide is an intron or exon region of mRNA.

[0090] In some implementations, the target is selected from the following group: EXOC2 Gene, Ku80 Genes and Task 1 Gene.

[0091] In some implementations, oligonucleotides will EXOC2 Gene, Ku80 Gene or Task 1 Gene expression is suppressed by at least approximately 50%.

[0092] In another aspect, this disclosure provides a method for treating or managing amyotrophic lateral sclerosis (ALS) in a patient, the method comprising administering to the patient a therapeutically effective amount of an oligonucleotide complementary to SOD1 as described herein.

[0093] In another aspect, this disclosure provides a method for treating or managing primary hyperlipidemia in a patient, the method comprising administering to the patient a therapeutically effective amount of dsRNA targeting PCSK9 as described herein.

[0094] In some implementation schemes, primary hyperlipidemia is defined as heterozygous familial hypercholesterolemia (HeFH).

[0095] In another aspect, this disclosure provides a method for reducing low-density lipoprotein cholesterol (LDL-C) in a patient, the method comprising administering to the patient a therapeutically effective amount of dsRNA targeting PCSK9 as described herein. Attached Figure Description

[0096] The foregoing and other features and advantages of this disclosure will be more fully understood through the following detailed description of illustrative embodiments, in conjunction with the accompanying drawings. This patent or application document contains at least one drawing shown in color. Upon request and payment of the necessary fees, the office will provide a copy of this patent or patent application publication with color drawings.

[0097] Figure 1 A schematic diagram illustrating an RNase H- and lysosomal knockdown method for knocking down target RNA.

[0098] Figure 2 A schematic diagram illustrating an exemplary Lyso-ASO that can target lysosomes. The spacer polymer ASO contains a 5' / 3' end 2' MOE modification and internal DNA nucleotides. Each nucleotide in the ASO is bonded with a phosphate thioester modified. The lyso sequence is unmodified.

[0099] Figure 3 Showing spacer polymers with and without polyG, polyA, or polyC ligands as measured by qRT-PCT analysis. EXOC2 mRNA levels. N = 7–9. Values ​​are mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, passed the two-tailed Steadon's t-test.

[0100] Figure 4 This demonstrates spacer polymers with and without poly-G ligands of different lengths, as measured by qRT-PCT analysis. EXOC2 mRNA levels. N = 6–12. Values ​​are mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, passed the two-tailed Steadon's t-test.

[0101] Figure 5 exhibit EXOC2 349. As measured by qRT-PCT analysis, relative EXOC2 mRNA levels. N = 3, independent differentiation. Values ​​are mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, passed the two-tailed Steudon's t test.

[0102] Figure 6 exhibit EXOC2 3933, as measured by qRT-PCT analysis, relative EXOC2 mRNA levels. N = 6. Values ​​are mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, passed by two-tailed Steadon's t-test.

[0103] Figure 7 A schematic diagram illustrating the effect of BAFILOMYCIN A1 (Baf A1) on lysosomal V-ATPase and thus on lysosomal function.

[0104] Figure 8 Demonstrating the effects of lysosomal inhibition and non-inhibition. EXOC2 3933, as measured by qRT-PCT analysis, relative EXOC2 mRNA levels. N = 6. Values ​​are mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, passed by two-tailed Steadon's t-test.

[0105] Figure 9 A schematic diagram illustrating an RNase H and lysosome-based knockdown method that uses spatially blocked ASO to knock down target RNA.

[0106] Figure 10 Exhibition space with and without barriers EXOC2 3933, as measured by qRT-PCT analysis, relative EXOC2 mRNA levels. N = 4–6. Values ​​are mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, passed the two-tailed Steadon's t-test.

[0107] Figure 11 exhibit Ku80 624 and 2802, as measured by qRT-PCT analysis, are relative Ku80 mRNA levels. N = 6. Values ​​are mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, passed by two-tailed Steadon's t-test.

[0108] Figure 12 exhibit TASK1 622 and 5968, as measured by qRT-PCT analysis, are relative values. TASK1 mRNA levels. N = 6. Values ​​are mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, passed by two-tailed Steadon's t-test.

[0109] Figure 13 The relative levels of SH-SY5Y cells treated with 25 or 100 nM non-targeted control (NTC), Tofersen, or Tofersen-LampASO for 24 hours, as measured by qRT-PCT analysis, are shown. SOD1 mRNA levels (n = 3). Values ​​are mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, passed by one-tailed t-test. Figure 14 The relative levels of cells treated with 50 nM inclisiran or inclisiran-Lamp for 48 hours are shown as measured by qRT-PCT analysis. PCSK9 mRNA levels (n = 4). Values ​​are mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, passed by one-tailed t-test.

[0110] Figure 15 The relative levels of SH-SY5Y cells treated for 30 hours with a mediator (simulant), 30 nM Tofsen-Lamp, or Tofsen-Lamp-derived ASO were shown as measured by qRT-PCT analysis. SOD1 mRNA levels (n = 3). Values ​​are mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, assessed by one-tailed or two-tailed t-test. Detailed Implementation

[0111] This disclosure provides oligonucleotides, methods, and compositions for degrading target polynucleotides (e.g., RNA) using the lysosomal pathway. The disclosure herein is largely based on the finding that antisense compounds conjugated to lysosomal ligands (e.g., poly-G sequences) facilitate the degradation of target polynucleotides via the lysosomal pathway. Without being bound by any particular theory, the lysosomal ligand from the antisense compound interacts with the LAMP-2C protein of the lysosome and triggers the lysosome's uptake and degradation of the target polynucleotide (e.g., antisense oligonucleotide) attached to the antisense compound.

[0112] It should be understood that the methods described in this disclosure are not limited to the specific methods and experimental conditions disclosed herein, as such methods and conditions can be modified. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0113] Furthermore, unless otherwise indicated, the experiments described herein may employ conventional molecular and cellular biological and immunological techniques within the scope of the art. Such techniques are well known to skilled technicians and are well explained in the literature. See, for example, Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987–2008) (including all supplements), MR Green and J. Sambrook, Molecular Cloning: A Laboratory Manual (4th ed.), and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd ed.).

[0114] Unless otherwise defined, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. Unless otherwise stated, the use of “or” means “and / or”. The use of the term “including” and other forms such as “includes” and “included” is not restrictive.

[0115] Generally, the nomenclature used in conjunction with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is that which is well-known and commonly used in the art. The nomenclature used in conjunction with the analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein, along with their laboratory procedures and techniques, is that which is well-known and commonly used in the art.

[0116] To make this disclosure easier to understand, some terms are first defined.

[0117] The term “nucleoside” refers to a molecule having a purine or pyrimidine base covalently linked to ribose or deoxyribose. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine, and thymidine. Other 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 a sugar moiety by an ester bond. Exemplary nucleotides include monophosphate, diphosphate, and triphosphate nucleosides. The terms “polynucleotide” and “nucleic acid molecule” are used interchangeably herein and refer to a polymer of nucleotides linked together by a phosphodiester or thiophosphate bond between the 5' and 3' carbon atoms.

[0118] The terms “RNA” or “RNA molecule” or “ribonucleic acid molecule” refer to a polymer 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” or “DNA molecule” or “deoxyribonucleic acid molecule” refer to a polymer of deoxyribonucleotides. A DNA nucleotide refers to a single deoxyribonucleotide. As used herein, the term “DNA-like” refers to, for example, a conformation of a modified nucleoside or nucleotide that resembles the conformation of the corresponding unmodified DNA unit. For example, a DNA-like nucleotide may refer to a modified deoxyribonucleotide that resembles the conformation of 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′-fluoroarabinonucleotide, also referred to in the art as 2′F-ANA or FANA), and corresponding phosphate thioester analogs. As used herein, the term "RNA-like" refers to a conformation, for example, of a modified nucleoside or nucleotide that resembles the conformation of the corresponding unmodified RNA unit. RNA-like conformations may employ an A-type helix, while DNA-like conformations employ 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 referred to in the art as 2′F-RNA), locked nucleic acid (LNA) nucleotides (also referred to in the art as bridging nucleic acids or bicyclic nucleotides), 2′-fluoro-4′-thioarabinonucleotides (also referred to in the art as 4′S-FANA nucleotides), 2′-O-alkyl-RNA, and corresponding thiophosphate analogs.

[0119] DNA and RNA can be naturally synthesized (e.g., through DNA replication or DNA transcription, respectively). RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized. 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 specifies the amino acid sequence of one or more polypeptide chains. This information is translated when the ribosome binds to the mRNA during protein synthesis.

[0120] As used herein, the term “small interfering RNA” (“siRNA”) (also referred to in the art as “short interfering RNA”) refers to an RNA (or RNA analog) containing about 10-50 nucleotides (or nucleotide analogs) capable of guiding or mediating RNA interference. In one embodiment, the siRNA contains about 15-30 nucleotides or nucleotide analogs, or about 16-25 nucleotides (or nucleotide analogs), or about 18-23 nucleotides (or nucleotide analogs), or about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21, or 22 nucleotides or nucleotide analogs). The term “short” siRNA refers to an siRNA containing about 21 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21, or 22 nucleotides). The term “long” siRNA refers to an siRNA containing about 24-25 nucleotides (e.g., 23, 24, 25, or 26 nucleotides). In some cases, short siRNAs may comprise fewer than 19 nucleotides, such as 16, 17, or 18 nucleotides, provided that the shorter siRNA retains the ability to mediate RNAi. Similarly, in some cases, long siRNAs may comprise more than 26 nucleotides, provided that the longer siRNA retains the ability to mediate RNAi without further processing (e.g., enzymatic processing) into short siRNAs.

[0121] The terms "nucleotide analogue," "altered nucleotide," or "modified nucleotide" refer to non-standard nucleotides, including ribonucleotides or deoxyribonucleotides that are not naturally occurring. Exemplary modified nucleotides are modified at any position to alter certain chemical properties of the nucleotide while retaining its ability to perform its intended function. Examples of derivatizable nucleotide positions include the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine, 5-propynyluridine, 5-propenyluridine, etc.; the 6-position, such as 6-(2-amino)propyluridine; and the 8-position of adenosine and / or guanosine, such as 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine, etc. Modified nucleotides also include denitronucleotides, such as 7-denitro-adenosine; O-modified and N-modified nucleotides (e.g., alkylation, such as N6-methyladenosine, or other nucleotides as known in the art) and other heterocyclic modified nucleotides, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., Aug 2000 10(4):297-310.

[0122] Modified nucleotides may 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. As another example, the ribose may be partially replaced by a bicyclic or tricyclic moiety, such as in locked nucleic acids, restricted ethyl groups, tricyclic DNA (tcDNA), or other bridging or bicyclic modifications. Other possible modifications include those described in U.S. Patent Nos. 5,858,988 and 6,291,438.

[0123] The phosphate group of a nucleotide can also be modified, for example, by replacing one or more oxygen atoms of the phosphate group with sulfur (e.g., thiophosphate), or by making other substitutions that allow the nucleotide to perform its intended function, as described in Eckstein, Antisense Nucleic Acid Drug Dev. April 2000 10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. October 2000 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. October 2001 11(5):317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. April 2001 11(2):77-85, and U.S. Patent No. 5,684,143. Some of the above modifications (e.g., phosphate group modifications) reduce the rate of hydrolysis of polynucleotides containing the like in vivo or in vitro.

[0124] As used herein, the term "unmodified nucleotide" or "non-modified nucleotide" refers to a nucleotide consisting of naturally occurring nucleobases, a sugar moiety, and nucleoside bonds. In some embodiments, the non-modified nucleotide is an RNA nucleotide (i.e., β-D-ribonucleoside) or a DNA nucleotide (i.e., β-D-deoxyribonucleoside).

[0125] The term "oligonucleotide" refers to a short polymer of nucleotides and / or modified nucleotides. As discussed above, oligonucleotides can be linked by non-phosphodiester bonds, resulting in a lower hydrolysis rate compared to oligonucleotides linked by phosphodiester bonds. For example, the nucleotides of an oligonucleotide may contain triazole, amide, carbamate, methylene glycol, ethylene glycol, oxymethylthio, oxyethylthio, oxycarbonyloxy, diaminophosphate, aminophosphate, phosphonate, and / or thiophosphate bonds. Modifications or alterations to oligonucleotides may also include adding non-nucleotide material to one or more ends or interiors (at one or more nucleotides of the oligonucleotide). Oligonucleotides may contain lysosomal ligands.

[0126] As used herein, the term "antisense oligonucleotide" refers to an oligonucleotide molecule capable of binding to intracellular RNA via Watson-Crick base pairing. Depending on the sequence and chemical properties of the antisense oligonucleotide, this interaction can cause either silencing of the target gene (i.e., reducing the expression level of mature mRNA and / or protein from that gene) or activation of the target gene (i.e., increasing the expression level of mature mRNA and / or protein from that gene). The antisense oligonucleotides of this disclosure focus on activating gene expression, which can be accomplished using various mechanisms. Some antisense oligonucleotides are designed to recruit RNase H to cleave their target RNA. RNase H is a family of non-sequence-specific endonucleases that catalyze the cleavage of RNA in RNA / DNA substrates via a hydrolytic mechanism. In some 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 is compatible with the activation of overall target gene expression (e.g., EXOC2, Ku80, and Task1 gene expression). Other antisense oligonucleotides (called steric blockers) are designed not to cause cleavage of their targets, but to block interactions with cytokines. For example, these cytokines can regulate splicing, block interactions between non-coding RNAs or RNA-binding proteins, stabilize mRNA to prolong its half-life, or increase the efficiency of mRNA translation.

[0127] Antisense oligonucleotides designed to recruit RNase H are typically designed as "spacer polymers." The term "spacer polymer" refers to a chimeric antisense oligonucleotide in which an inner region containing multiple nucleotides supporting RNase H cleavage is situated between an outer region containing one or more nucleotides, wherein the nucleotides constituting the inner region are chemically distinct from the one or more nucleotides constituting the outer region. The inner region may be called a "spacer segment," and the outer region may be called a "wing segment." A "chimeric antisense oligonucleotide" is an antisense oligonucleotide having at least two chemically distinct regions.

[0128] As used herein, the term "lysosomal ligand" is a ligand that interacts with a lysosome. Unbound by any particular theory, the lysosomal ligand interacts with the LAMP-2C protein of the lysosome and triggers the uptake and degradation of target RNA linked to an oligonucleotide containing the lysosomal ligand. In some embodiments, the lysosomal ligand is a poly-G ligand or sequence.

[0129] As used herein, a “poly-G ligand” or “poly-G sequence” is a polynucleotide sequence containing two or more G nucleotides and lacking complementarity with the target polynucleotide of the oligonucleotide linked to the poly-G sequence.

[0130] In some embodiments, the poly-G sequence comprises 2-30 G nucleotides. In some embodiments, the poly-G sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 5 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 6 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 7 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 8 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 9 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 10 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 11 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 12 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 13 G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 14 G nucleotides. In some implementations, the polyG sequence comprises or consists of 15 G nucleotides.

[0131] In some embodiments, the polyG sequence is linked to the 3' end of the oligonucleotide of this disclosure. In other embodiments, the polyG sequence is linked to the 5' end of the oligonucleotide of this disclosure. In other embodiments, the polyG sequence is linked to both the 5' and 3' ends of the oligonucleotide of this disclosure.

[0132] In some implementations, the polyG sequence is single-stranded.

[0133] In some embodiments, the polyG sequence binds to proteins on the surface of the lysosome. In some embodiments, the polyG sequence binds to a lysosome-associated membrane glycoprotein (LAMP). In some embodiments, the LAMP is LAMP2C.

[0134] In some embodiments, the poly-G sequence comprises consecutive G nucleotides. In some embodiments, the poly-G sequence comprises or consists of 5-15 consecutive G nucleotides. In some embodiments, the poly-G sequence comprises one or more non-G nucleotides (e.g., A, T, U, or C) within the poly-G sequence. In some embodiments, the oligonucleotide comprises two or more poly-G sequences, with one or more non-G nucleotides (e.g., A, T, U, or C) between the two or more poly-G sequences. For example, but by no means limiting, the oligonucleotide may comprise a first poly-G sequence of 5-10 consecutive G nucleotides, one or more non-G nucleotides (e.g., A, T, U, or C), and a second poly-G sequence of 5-10 consecutive G nucleotides at its 3' end (from 5' to 3').

[0135] In some implementations, the poly-G sequence comprises discontinuous G nucleotides.

[0136] In some embodiments, the polyG sequence comprises or consists of (dG)(dG)(dG)(dG)(dG)(dG). (This text is repeated 6 times in the original.) In some embodiments, the polyG sequence comprises or is composed of (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dG)(dN)(dG), wherein each dN individually corresponds to a deoxyribonucleotide of dA, dT, or dC. In some embodiments, the polyG sequence comprises or is composed of (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dG)(dN)(dG)(dG), wherein each dN individually corresponds to a deoxyribonucleotide of dA, dT, or dC. In some embodiments, the polyG sequence comprises or is composed of (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dG)(dN)(dG)(dG), wherein each dN individually corresponds to a deoxyribonucleotide of dA, dT, or dC.

[0137] As used herein, the term "functional moiety" is the portion linked to an oligonucleotide. The functional moiety is linked to the 5' or 3' end of the oligonucleotide. The functional moiety may comprise an N-acetylgalactosamine (GalNAc) moiety and / or a hydrophobic moiety. The hydrophobic moiety may be a fatty acid, steroid, open-ring steroid, lipid, ganglioside, nucleoside analog, endocannabinoid, vitamin, or mixtures thereof. Steroids may be cholesterol and lithocholic acid (LCA). Fatty acids may be eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanoic acid (DCA).

[0138] As used herein, the term "target polynucleotide" refers to a polynucleotide that is sufficiently complementary to the oligonucleotides of this disclosure to mediate lysosomal targeting via a polyG sequence. Exemplary target polynucleotides include mRNA and viral RNA.

[0139] As used herein, the term "target gene" refers to a gene whose expression will be substantially silenced, restored, or increased. In some embodiments, target gene expression is silenced, restored, or increased to wild-type levels via oligonucleotides that interact with the target sequence through direct base pairing and via lysosomal pathways (e.g., EXOC2, Ku80, and Task1 mRNA target sequences). In some embodiments, target gene expression is silenced, restored, or increased to wild-type levels via RNA silencing (e.g., by cleaving transcripts corresponding to the target gene or by translational repression of the target gene). Without wishing to be bound by theory, cleavage of the target transcript can increase the productive transcriptional level of the target gene. For example, but by no means limited, alleles of the target gene can be expressed to form premRNA, which can be defective (e.g., containing nucleotide repeat regions that cause disease). Target gene expression can be restored by cleaving and degrading defective premRNA derived from defective alleles, thereby releasing transcriptional mechanisms to trigger transcription of non-defective target gene alleles. The term "non-target gene" refers to a gene whose expression is not significantly increased, restored, or silenced. For example, the target genes of this disclosure are EXOC2, Ku80, and Task1, while the non-target genes of this disclosure are genes that are not EXOC2, Ku80, and Task1. In one embodiment, the polynucleotide sequences (e.g., mRNA encoded by the target and non-target genes) may differ by one or more nucleotides. In another embodiment, the target and non-target genes may differ due to one or more polymorphisms (e.g., single nucleotide polymorphisms or SNPs). In another embodiment, the target and non-target genes may have less than 100% sequence identity. In another embodiment, the target and non-target genes may have 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 of the target gene (e.g., an ortholog or paralog).

[0140] The term "antisense activity" refers to any detectable or measurable activity attributable to the hybridization of an antisense compound with 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 a target nucleic acid. "Antisense compound" refers to an oligomeric compound capable of hybridizing with a target nucleic acid via hydrogen bonding. As used herein, "antisense oligonucleotide" refers to a single-stranded oligonucleotide having a nucleobase sequence that allows hybridization with a corresponding region or segment of the target nucleic acid.

[0141] The term "antisense inhibition" refers to a reduction in the level of the target nucleic acid in the presence of an antisense compound compared to the level in the absence of an antisense oligonucleotide having a sequence sufficiently complementary to the target nucleic acid. The target nucleic acid can be any nucleic acid. For example, the target nucleic acid of this disclosure can be EXOC2, Ku80, Task1, SOD1, or PCSK9 transcripts. In some embodiments, the target nucleic acid is EXOC2, Ku80, Task1, SOD1, or PCSK9 pre-mRNA.

[0142] The term "target recognition sequence" refers to the portion of an antisense compound that recognizes the target nucleic acid. A target recognition sequence has a nucleobase sequence that allows hybridization with the corresponding region or segment of the target nucleic acid.

[0143] The term "conserved region" refers to one or more conserved portions of a nucleic acid sequence, that is, one or more portions of a nucleic acid sequence that have similar or identical sequences across species. Conserved regions can be identified computationally, for example, using any sequence alignment software available in the art.

[0144] As used herein, the term "fully complementary" means that the antisense compound has a sequence (e.g., an antisense oligonucleotide with a target recognition sequence) sufficient to bind to the desired target transcript (e.g., EXOC2, Ku80, or Task1 transcript) and 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 the EXOC2, Ku80, or Task1 transcript) can be considered sufficiently complementary to increase, restore, or silence the expression of the EXOC2, Ku80, or Task1 gene. The term "perfectly complementary" means, for example, that the target recognition sequence is 100% complementary to the target nucleic acid sequence. Complementary nucleic acid molecules hybridize with each other. The term "hybridization" refers to the annealing of complementary nucleic acid molecules. In some embodiments, the complementary nucleic acid molecule comprises an antisense compound and a target nucleic acid.

[0145] As used herein, "complementary region" refers to the portion of an antisense oligonucleotide that is complementary to the target transcript (e.g., EXOC2, Ku80, or Task1 transcripts). For example, but by no means limiting, an 18-nucleotide-long antisense oligonucleotide may contain a continuous 12-nucleotide portion complementary to the target transcript. In some embodiments, the antisense oligonucleotide is complementary to the target transcript along its entire length.

[0146] As used herein, “administer / administration” means the act of physically delivering a substance (such as the antisense compounds described herein) into a patient by injection or other means while it is present outside the body. The antisense oligonucleotides described herein can be administered to a patient’s central nervous system. 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 striatal infusion or delivery and / or any other physical delivery method described herein or known in the art. Intraventricular infusion may include administration using an Ommaya reservoir. In some embodiments, the antisense oligonucleotides described herein can be administered to a patient systemically (e.g., intravenously, subcutaneously, or intramuscularly). These compounds may be engineered to enter the central nervous system or to be active in other tissues, such as muscles (including skeletal or cardiac muscle) or the pancreas.

[0147] When managing or treating a disease or its symptoms, the administration of a substance typically occurs after the onset of the disease or its symptoms. When preventing a disease or its symptoms, the administration of a substance typically occurs before the onset of the disease or its symptoms and can continue for a long period to delay the appearance of disease-related symptoms (such as damage to related tissues and the respiratory tract) or reduce their severity.

[0148] As used herein, the term "composition" is intended to cover products containing optionally specified amounts of a particular ingredient (such as the antisense compound provided herein), and any product produced directly or indirectly from a combination of optionally specified amounts of the specified ingredients.

[0149] "Effective amount" refers to the amount of an active pharmaceutical agent (such as the antisense compound of this disclosure) sufficient to achieve the desired physiological outcome in an individual who requires the agent. Effective amounts can vary between individuals depending on the health and physical condition of the individual to be treated, the individual's taxonomy, the formulation of the composition, the assessment of the individual's medical condition, and other relevant factors.

[0150] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject may be a mammal, such as a non-primate (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In some 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 livestock, farm animals, sporting animals, and pets. In one embodiment, a subject is a patient suffering from... EXOC2 Related symptoms Ku80 Related conditions and / or Task 1 Mammals with related conditions, such as humans. In another implementation, the subject is a developing... EXOC2 Related symptoms Ku80Related conditions and / or Task 1 Mammals, such as humans, are at risk of developing related diseases.

[0151] As used herein, the term "therapy" refers to any treatment that can be used to prevent, manage, treat, and / or improve a disease or its associated symptoms (such as...). EXOC2 Related symptoms Ku80 Related conditions and / or Task 1 Any regimen, method, and / or agent that can be used to modulate a subject's immune response to an infection or related symptoms. In some embodiments, the term "therapy" refers to any regimen, method, and / or agent that can be used to modulate a subject's immune response to an infection or related symptoms. In some embodiments, the term "one or more therapies" refers to biological therapies, supportive therapies, and / or those known to those skilled in the art (such as medical personnel) for the prevention, management, treatment, and / or improvement of a disease or related symptoms (such as...). EXOC2 Related symptoms Ku80 Related conditions and / or Task 1 Other treatments for (related conditions). In other embodiments, the term "one or more therapies" means biological therapies, supportive therapies, and / or other therapies known to those skilled in the art that can be used to modulate a subject's immune response to infection or related symptoms.

[0152] As used herein, the term "treat / treatment / treating" refers to a disease or related symptoms (such as those caused by the application of one or more therapies (including, but not limited to, the application of one or more preventative or therapeutic agents, such as the antisense oligonucleotides provided herein)). EXOC2 Related symptoms Ku80 Related conditions and / or Task 1 The reduction or improvement of the progression, severity, and / or duration of the associated disease. As used herein, the term "treatment" may also refer to alterations to the course of the disease in the treated subject. The therapeutic effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of one or more symptoms, reduction of the direct or indirect pathological consequences of the disease, reduction of the rate of disease progression, improvement or mitigation of the disease state, and relief or improvement of prognosis.

[0153] As used herein, the term "EXOC2" refers to the gene encoding component 2 of the protein exocapsule complex. The protein EXOC2 is a component of the exocapsule complex, a multi-protein complex essential for the polarization of exocapsids to specific docking sites on the plasma membrane. The human EXOC2 gene corresponds to NG_047166.1 in the NCBI RefSeq database.

[0154] As used herein, the term "Ku80" refers to the protein encoded by the XRCC5 gene. Ku80 is a component of a heterodimer of Ku and Ku70, which 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 both the gene encoding Ku80 and its mRNA. Therefore, oligonucleotides targeting Ku80 target Ku80 mRNA (including pre-mRNA).

[0155] As used herein, the terms “Task1” or “KCNK3” refer to the gene encoding a member 3 protein of the potassium channel subfamily K. Task1 is a member of the potassium channel protein superfamily and contains two pore-forming P domains. The human KCNK3 gene corresponds to NG_033884.1 in the NCBI RefSeq database. Inhibitors of SOD1 (such as inxile) are used to treat hyperlipidemia or lower low-density lipoprotein cholesterol (LDL-C) in subjects.

[0156] As used herein, the term “SOD1” refers to the gene encoding superoxide dismutase 1 (SOD1) protein. SOD1 is a mammalian enzyme that catalyzes the removal of superoxide groups. The human SOD1 gene corresponds to NG_008689.1 in the NCBI RefSeq database. Inhibitors of SOD1 (such as toffson) are used to treat subjects with amyotrophic lateral sclerosis (ALS).

[0157] As used herein, the term "PCSK9" refers to the gene encoding the proprotein convertase subtilisin / kexin type 9 protein. PCSK9 binds to receptors on low-density lipoprotein (LDL) particles and degrades them. The human PCSK9 gene corresponds to NG_009061.1 in the NCBI RefSeq database. Inhibitors of PCSK9 (such as inxilex) are used to treat hyperlipidemia or lower LDL-C in subjects.

[0158] Antisense compounds This disclosure provides an antisense compound linked to the polyG sequence disclosed herein for directing target polynucleotides to lysosomes of cells for target degradation.

[0159] This disclosure also provides an antisense compound, which is capable of enabling... EXOC2 , Ku80 as well as Task 1Gene expression decreased 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.

[0160] In some implementations, it is possible to reduce the expression of target polynucleotides (e.g. EXOC2 , Ku80 as well as Task 1 Antisense compounds (gene expression) have chemically modified subunits arranged in various patterns or motifs, thereby endowing antisense compounds with properties such as enhanced activity, increased binding affinity to target nucleic acids, or resistance to degradation by nucleases in vivo.

[0161] In some embodiments, the antisense compounds of this disclosure are antisense oligonucleotides. Chimeric antisense oligonucleotides typically contain at least one modified region to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity to target nucleic acids, and / or increased activity. A second region of the chimeric antisense compound may optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of the RNA:DNA duplex. In some embodiments, the antisense compounds of this disclosure are chimeric antisense oligonucleotides having a spacer polymer motif. In the spacer polymer, an internal region having multiple nucleotides supporting RNase H cleavage is located between external regions having multiple nucleotides nucleotides chemistryically different from the internal region.

[0162] In some embodiments, this disclosure provides an antisense oligonucleotide having a target recognition sequence, the target recognition sequence being similar to... EXOC2 , Ku80 and / or Task 1 The transcript or a portion thereof is fully complementary, thereby guiding RNase H to... EXOC2 , Ku80 as well as Task 1 Transcription cleavage. The target recognition sequence of the antisense oligonucleotide can be the full length of the antisense oligonucleotide or a portion thereof. In some embodiments, the antisense oligonucleotide contains a spacer polymer motif.

[0163] In the case of antisense compounds with spacer polymer motifs, the spacer segment typically acts as a substrate for endonuclease cleavage, while the wing segment contains a modified nucleoside. In some embodiments, the regions of the spacer polymer are distinguished according to the type of sugar moiety comprising each distinct region. In some embodiments, the types of sugar moiety used to distinguish the regions of the spacer polymer may include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOE and 2′-O-CH3 (i.e., OMe) etc.), and bicyclic sugar-modified nucleosides (such bicyclic sugar-modified nucleosides may include those with 4′-(CH2)). n Nucleosides modified with bicyclic sugars of the -O-2′ bridge, where n=1 or n=2. In some embodiments, the wing segments of the spacer polymer contain one or more tricyclic-DNA (tcDNA) modifications. In some embodiments, each distinct region contains a homogeneous 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 combined for an antisense compound. In one embodiment, LNA modification and 2′-O-methyl modification can be combined for an antisense compound. In one embodiment, LNA modification and 2′-deoxy modification can be combined for an antisense compound. In one embodiment, LNA modification and tricyclic-DNA modification can be combined for an antisense compound. In one embodiment, 2′-MOE modification and tricyclic-DNA modification can be combined for an antisense compound.

[0164] The spacer polymer motif can be described using the formula "ABC", where "A" represents the length of the 5' wing region, "B" represents the length of the spacer region, and "C" represents the length of the 3' wing region. Therefore, in some embodiments, the antisense oligonucleotide of this disclosure has the following formula: ABC.

[0165] As used herein, the spacer polymer described as "ABC" has a configuration in which the spacer segment is positioned immediately adjacent to each of the 5' wing segment and the 3' wing segment. Therefore, there is no intermediate nucleotide between the 5' wing segment and the spacer segment or between the spacer segment and the 3' wing segment.

[0166] In some embodiments, the 5' wing region represented by "A" contains about 0 to about 8 modified nucleotides, such as about 1 to about 6 modified nucleotides. For example, the length of the 5' wing region represented by "A" can be 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, such as about 1 to about 6 modified nucleotides. For example, the length of the 3' wing region represented by "C" can be 0, 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides. In some embodiments, "A" and "C" are the same; in other embodiments, they are different.

[0167] In some embodiments, the spacer region represented by "B" comprises 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 length of the spacer region represented by "B" may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 DNA nucleotides and / or DNA-like nucleotides. Therefore, the antisense oligonucleotides of this disclosure having a target recognition sequence of formula "ABC" include, but are not limited to, the following spacer aggregate patterns, such as 1-10-1. (i.e., one nucleotide - ten nucleotides - one 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, 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 may also have different lengths, such as 1-10-6, 3-9-5, 7-9-2, 4-10-5, or other asymmetric combinations of wing lengths adjacent to the central DNA spacer. In some embodiments, the spacer aggregate of “ABC” is at least 12 nucleotides long. In some embodiments, the length of “B” is at least 6 nucleotides. Those skilled in the art will be able to identify other asymmetric combinations of wing lengths.

[0168] In some embodiments, the antisense compound targeting EXOC2, Ku80, or Task1 nucleic acids has a 5-9-4 spacer polymer pattern. In some embodiments, the antisense compound is an antisense oligonucleotide with a target recognition sequence having a 5-9-4 pattern, said target recognition sequence being sufficiently complementary to the EXOC2, Ku80, or Task1 transcript or a portion thereof to guide RNase H cleavage of the EXOC2, Ku80, or Task1 transcript. In some embodiments, the target recognition sequence has the formula "ABC", wherein "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", wherein "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", wherein "A" comprises 2 to 6 2'- O -(2-methoxyethyl) (MOE) modified nucleotides, where "B" contains 6 to 12 DNA nucleotides and / or DNA-like nucleotides, and "C" contains 2 to 6 2'- O -(2-methoxyethyl) (MOE) modified nucleotides. In some embodiments, the target recognition sequence has the formula "ABC", where "A" contains five 2'- O -(2-methoxyethyl) (MOE) modified nucleotides, where "B" contains 9 DNA nucleotides and / or DNA-like nucleotides, and "C" contains 4 2'- O -(2-methoxyethyl) (MOE) modified nucleotides.

[0169] In some embodiments, the 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 spacer region. Therefore, in some embodiments, the antisense oligonucleotides of this disclosure have the following formula: XY, or YX.

[0170] As used herein, winged polymers described as “XY” or “YX” have a configuration in which the spacer segment is positioned immediately adjacent to the wing segment. Therefore, there is no intermediate nucleotide between the wing segment and the spacer segment. Winged polymer configurations of the antisense compounds disclosed herein 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.

[0171] In some embodiments, antisense compounds targeting EXOC2, KU80, or TASK1 nucleic acids have spaced-widened motifs. As used herein, "spaced-widened" refers to an antisense compound having a spacer segment of 12 or more consecutive DNA nucleotides and / or DNA-like nucleotides adjacent to a wing region. In the case of spaced-widened spacer aggregates, the spacer aggregate comprises a spacer region of 12 or more consecutive DNA nucleotides and / or DNA-like nucleotides located between and immediately adjacent to the 5' and 3' wing segments. In the case of spaced-widened wing aggregates, the wing aggregate comprises a spacer region of 12 or more consecutive DNA nucleotides and / or DNA-like nucleotides located immediately adjacent to the wing segment.

[0172] Nucleosides are base-sugar combinations. The nucleobase (also called the base) moiety of a nucleoside is usually a heterocyclic base moiety. A nucleotide is a nucleoside that also includes a phosphate group covalently linked to the sugar moiety of the nucleoside. For those nucleosides that include pentofuranosyl sugars, the phosphate group may be linked to the 2′, 3′, or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed by covalently linking adjacent nucleosides to each other to form linear polymeric oligonucleotides. Within the oligonucleotide structure, the phosphate group is generally considered to form the nucleoside internucleotide bond of the oligonucleotide.

[0173] Modification of antisense compounds encompasses the substitution or alteration of nucleoside internucleotide bonds, sugar moieties, or nucleobases. Modified antisense compounds often outperform their natural counterparts due to desired properties such as enhanced cellular uptake, increased affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity.

[0174] Chemically modified nucleosides can also be used to increase the binding affinity of shortened or truncated antisense oligonucleotides to their target nucleic acids. Therefore, similar results can often be obtained using shorter antisense compounds with such chemically modified nucleosides.

[0175] Naturally occurring RNA and DNA have 3′ to 5′ phosphodiester bonds. Due to desired properties such as enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases, antisense compounds with one or more modified (i.e., non-naturally occurring) nucleotide bonds are often chosen over antisense compounds with naturally occurring nucleotide bonds.

[0176] Oligonucleotides with modified nucleoside bonds include those retaining a phosphorus atom and those without a phosphorus atom. Representative phosphorus-containing nucleoside bonds include, but are not limited to, phosphate diesters, phosphate triesters, methylphosphonates, aminophosphates, and thiophosphates. Methods for preparing phosphorus-containing and phosphorus-free oligonucleotides are well known.

[0177] In some embodiments, the antisense compound targeting EXOC2, KU80, or TASK1 nucleic acids comprises one or more modified nucleoside internucleotide bonds. In some embodiments, the modified nucleoside internucleotide bonds are phosphate thioester bonds. In some embodiments, each nucleoside internucleotide bond of the antisense compound is a phosphate thioester nucleoside internucleotide bond.

[0178] The antisense compounds of this disclosure may optionally contain one or more nucleosides in which the sugar groups have been modified. Such sugar-modified nucleosides may confer enhanced nuclease stability, increased binding affinity, or other beneficial biological properties to the antisense compounds. In some embodiments, the nucleoside comprises a chemically modified furanose ring moiety. Examples of chemically modified furanose rings include, but are not limited to, the addition of substituents (including 5′ and 2′ substituents, bridging ring atoms to form bicyclic nucleic acids (BNAs), and the use of S, N(R), or C(R) groups. 1 (R) 2 (R=H, C1-C) 12 Alkyl or protecting groups) replace the ribosyl epoxy atom and combinations thereof. Examples of chemically modified sugars include 2′-F-5′-methyl substituted nucleosides (see other 5′,2′-disubstituted nucleosides disclosed in PCT International Application WO 2008 / 101157, published August 21, 2008) or ribosyl epoxy atoms substituted with S and further substituted at the 2′-position (see published U.S. Patent Application US2005-0130923, published June 16, 2005) or 5′-substituted BNA (see PCT International Application WO 2007 / 134181, published November 22, 2007, wherein LNA is substituted with, for example, 5′-methyl or 5′-vinyl).

[0179] Examples of nucleosides with modified sugar moieties include, but are not limited to, nucleosides containing 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. The substituent at the 2′ position may also be selected from 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 ), where each R m and R n Independently, it is H or a substituted or unsubstituted C1-C10 alkyl group. 2'-Modified nucleotides can be used in this disclosure, such as 2'-O-methylRNA, 2'-O-methoxyethylRNA, 2'-fluoroRNA, and other 2'-modified nucleotides contemplated by those skilled in the art.

[0180] Examples of bicyclic nucleic acids (BNAs) include, but are not limited to, nucleosides containing bridges between 4′ and 2′ ribosyl ring atoms. BNAs containing bridges between 4′ and 2′ ribosyl ring atoms may be referred to as locked nucleic acids (LNAs) and are often referred to as inaccessible RNA. As used herein, the term “locked nucleotide” or “locked nucleic acid (LNA)” includes a nucleotide in which the 2′ deoxyribose moiety is modified by introducing a structure containing heteroatoms bridging from the 2′ to 4′ carbon atoms. The term “non-locked nucleotide” includes nucleotides that do not contain bridging structures in the ribose moiety. Therefore, the term encompasses 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-pentafuranosyl or ribose-pentafuranosyl moiety. In some embodiments, the antisense compounds provided herein comprise one or more BNA nucleosides, wherein the bridge comprises one of the following formulas: 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-N(OCH3)-2′ (see PCT / US2008 / 064591); 4′-CH2-ON(CH3)-2′ (See published U.S. Patent Application US2004-0171570, published September 2, 2004); 4′-CH2-N(R)-O-2′ (see U.S. Patent No. 7,427,672, published September 23, 2008); 4′-CH2-C(CH3)-2′ and 4′-CH2-C(=CH2)-2′ (see PCT / US2008 / 066154); and wherein R is independently H, C1-C12 alkyl or protecting group. The aforementioned BNAs include various stereochemical sugar configurations, including, for example, α-L-ribofranose and β-D-ribofranose (see PCT International Application PCT / DK98 / 00393, published March 25, 1999 as WO 99 / 14226).

[0181] In some embodiments, the antisense compounds provided herein comprise one or more 2',4'-restricted nucleotides. For example, the antisense compounds provided herein include those having 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.

[0182] In some embodiments, the nucleoside is modified by replacing the ribosyl ring with a sugar substitute. Such modifications include, but are not limited to, replacing the ribosyl ring with a substitute ring system (sometimes called a DNA analogue), such as a morpholine ring, a cyclohexenyl ring, a cyclohexyl ring, or a tetrahydropyranyl ring, such as a tetrahydropyranyl ring having one of the following formulas: , or

[0183] In some embodiments, the antisense oligonucleotide may comprise a morpholine ring linked by a diaminophosphate bond. These may be referred to as PMO oligomers or diaminophosphate morpholine oligomers. In some such embodiments, the backbone of these oligonucleotides may be uncharged. In other embodiments, one or more of the diaminophosphate bonds may comprise a charged portion.

[0184] Many other bicyclic and tricyclic sugar substitute ring systems are also known in the art and can be used to modify nucleosides for incorporation into antisense compounds (see, for example, review article: Leumann, J. C, Bioorganic & Medicinal Chemistry , 2002, 10, 841-854; Ito, KR; Obika, S., Recent Advances in Medicinal Chemistry of Antisense Oligonucleotides. Comprehensive Medicinal Chemistry, 3rd Edition (Elsevier: 2017). Such ring systems can undergo various other substitutions to enhance activity.

[0185] Methods for preparing modified sugars are well known to those skilled in the art. In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified, or a combination thereof) retain hybridization with appropriate nucleic acid targets.

[0186] In some embodiments, the antisense compound targeting EXOC2, Ku80, or Task1 nucleic acids comprises one or more modified nucleotides. In one embodiment, the antisense compound targeting EXOC2, Ku80, or Task1 nucleic acids comprises a 2'-modified nucleotide. In one embodiment, the antisense compound targeting EXOC2, Ku80, or Task1 nucleic acids comprises 2'-O-methylRNA, 2'-O-methoxyethylRNA, or 2'-fluoroRNA. In one embodiment, the antisense compound targeting EXOC2, Ku80, or Task1 nucleic acids comprises tricyclic-DNA (tcDNA). Tricyclic-DNA belongs to a class of constrained DNA analogs that exhibit improved hybridization ability with 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, the antisense compound targeting EXOC2, Ku80, or Task1 nucleic acids comprises a locked nucleotide, an ethyl-restricted nucleotide, or an α-L-locked nucleic acid. Various α-L-locked nucleic acids are known to those skilled in the art, and are, for example, found in Sorensen et al. J. Am. Chem. Soc. It is described in (2002) 124(10):2164-2176.

[0187] In some embodiments, the antisense compounds targeting EXOC2, Ku80, or Task1 nucleic acids are fully chemically modified, meaning that each nucleotide is chemically modified. In some embodiments, each nucleotide contains a 2'- O -(2-methoxyethyl) (MOE) modification. In some embodiments, each nucleotide contains a tricyclic-DNA modification. In some embodiments, the antisense compound targeting EXOC2, Ku80, or Task1 nucleic acids contains a tricyclic-DNA modification and a 2'- O A mixture modified with -(2-methoxyethyl) (MOE), wherein each nucleotide of the antisense compound is either tcDNA or MOE.

[0188] In some embodiments, the antisense compound targeting EXOC2, Ku80, or Task1 nucleic acids comprises one or more modified nucleotides having a modified sugar moiety. In some embodiments, the modified nucleotides are locked nucleotides. In some embodiments, the locked nucleotides are arranged in a spacer polymer motif, such as a 3-9-3 spacer polymer pattern, wherein nine non-locked nucleotides are side-mounted with three locked nucleotides on each side.

[0189] Nucleobase (or base) modifications or substitutions are structurally distinguishable from naturally occurring or synthetically unmodified nucleobases, but functionally interchangeable. Both natural and modified nucleobases can participate in hydrogen bonding. Such nucleobase modifications can confer antisense compounds with nuclease stability, binding affinity, or other beneficial biological properties. Modified nucleobases include synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitution, can be used to increase the binding affinity of antisense compounds to target nucleic acids. For example, 5-methylcytosine substitution has been shown to increase the stability of nucleic acid duplexes by 0.6–1.2 °C (Sanghvi, YS, Crooke, ST, and Lebleu, B. eds.). Antisense Research and Applications (CRC Press, Boca Raton, 1993, pp. 276-278).

[0190] Other modified nucleobases include 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, adenine, and guanine's 6-methyl and other alkyl derivatives, adenine and guanine's 2-propyl and other alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3)uracil and cytosine, and other alkynyl derivatives of pyrimidine bases; 6-azouracil, cytosine, and thymine; 5-uracil. (Pseudouracil); 4-Thiouracil; 8-halogen, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine; 5-halogen (such as 5-bromine), 5-trifluoromethyl and other 5-substituted uracil and cytosine; 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deadenine and 7-deadenine, and 3-deadenine and 3-deadenine.

[0191] The heterocyclic base moiety may also include those heterocyclic base moietyes in which the purine or pyrimidine base is substituted with other heterocyclic compounds, such as 7-deadenine, 7-deadenine, 2-aminopyridine, and 2-pyridone. Nucleobases that can be used to increase 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.

[0192] In some embodiments, the antisense compound targeting EXOC2, Ku80, or Task1 nucleic acids comprises one or more modified nucleotides having a modified sugar moiety. In some embodiments, the modified nucleotide is a locked nucleotide. In some embodiments, the locked nucleotides are arranged in a spacer polymer motif, such as a 3-9-3 spacer polymer pattern, wherein nine non-locked nucleotides are side-mounted with three locked nucleotides on each side. In some embodiments, the antisense compound targeting EXOC2, Ku80, or Task1 nucleic acids comprises one or more modified nucleotides. In some embodiments, the modified nucleotide is 5-methylcytosine. In some embodiments, each cytosine is 5-methylcytosine. In some embodiments, the modified nucleotide is 2'- O -(2-methoxyethyl) (MOE) modified nucleotides. In some embodiments, 2'- O -(MOE) modified nucleotides are arranged in spacer polymer motifs, such as the 5-9-4 spacer polymer pattern, in which 9 non-2'- O -(MOE) modified nucleotides are flanked by 4 or 5 2'- nucleotides on one or both sides. O -(MOE) modified nucleotides. In some embodiments, the antisense compound targeting EXOC2, Ku80, or Task1 nucleic acids comprises a sterically blocking chemical modification pattern. In some embodiments of the sterically blocking chemical modification pattern, each nucleotide of the antisense compound is 2'- O -(2-methoxyethyl) (MOE) modified nucleotides. In some embodiments of the sterically blocking chemical modification mode, each nucleotide of the antisense compound is a tricyclic-DNA modified nucleotide. In some embodiments of the sterically blocking chemical modification mode, the antisense compound comprises at least one MOE modified nucleotide and at least one tricyclic-DNA modified nucleotide. Many different chemical modification modes for sterically blocking antisense oligonucleotides are envisioned. For example, but by no means limited, sterically blocking antisense oligonucleotides may comprise mixtures of different types of modifications, such as 2'- O -(2-methoxyethyl) modification, LNA modification, tricyclic-DNA modification, and mixtures of DNA modifications, wherein the DNA extension is four nucleotides or less.

[0193] In some embodiments, the antisense compound of this disclosure directs RNase H to cleave the EXOC2, Ku80, or Task1 transcripts. In such embodiments, the antisense compound may be referred to as an RNase H-dependent antisense compound. In some embodiments, the antisense compound is an RNase H-dependent antisense oligonucleotide. In some embodiments, the antisense oligonucleotide of this disclosure is an RNase H-dependent antisense oligonucleotide and may be a single-stranded, chemically modified oligonucleotide that binds to a complementary sequence in the target transcript (e.g., the EXOC2, Ku80, or Task1 transcript). The RNase H-dependent antisense oligonucleotide of this disclosure reduces the expression of the target gene by RNase H-mediated target transcript cleavage and by inhibiting translation through ribosomal steric hindrance. In some embodiments, the antisense compound of this disclosure is capable of mediating 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 RNase-H cleavage of EXOC2, Ku80 or Task1 transcripts. In one embodiment, the antisense compound is capable of mediating at least 80% of the RNase-H cleavage of EXOC2, Ku80 or Task1 transcripts. In one embodiment, the antisense compound is capable of mediating at least 90% of the RNase-H cleavage of EXOC2, Ku80 or Task1 transcripts.

[0194] In some embodiments, the antisense compound targeting the EXOC2, Ku80, or Task1 transcripts is about 6 to about 24 subunits in length. In other embodiments, the antisense compound targeting the EXOC2, Ku80, or Task1 transcripts is about 8 to about 80 subunits in length. For example, the length of the antisense compound is 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 connected subunits or a range determined by any two of the above values. In some embodiments, the antisense compound is less than 40 linked subunits in length. In some embodiments, the antisense compound is about 10 to about 30 linked subunits in length. In some embodiments, the antisense compound is about 12 to about 25 linked subunits in length. In some embodiments, the antisense compound is about 15 to about 20 linked subunits in length. In some embodiments, the antisense compound is an antisense oligonucleotide targeting EXOC2, Ku80, or Task1 transcripts, and the linked subunits are linked nucleotides.

[0195] In some implementations, antisense compounds targeting EXOC2, Ku80, or Task1 transcripts may be shortened or truncated. For example, a single subunit may be deleted from the 5′ end (5′ truncated) or from the 3′ end (3′ truncated). Shortened or truncated antisense compounds targeting EXOC2, Ku80, or Task1 transcripts may delete two subunits from the 5′ end of the antisense compound, or two subunits from the 3′ end. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound that deletes one nucleoside from the 5′ end and one nucleoside from the 3′ end.

[0196] When a single additional subunit is present in an extended antisense compound, the additional subunit may be located at the 5′ or 3′ end of the antisense compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, an antisense compound in which two subunits are added to either the 5′ end (5′ addition) or the 3′ end (3′ addition). Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, an antisense compound in which one subunit is added to the 5′ end and one subunit is added to the 3′ end.

[0197] It is possible to increase or decrease the length of antisense compounds (such as antisense oligonucleotides) and / or introduce mismatched bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), the ability of a series of antisense oligonucleotides of 13–25 nucleotides in length to induce target RNA cleavage was tested in an oocyte injection model. Antisense oligonucleotides of 25 nucleotides in length with 8 or 11 mismatched bases near the end of the antisense oligonucleotide were able to guide specific cleavage of the target mRNA, albeit to a lesser extent than those without mismatches. Similarly, target-specific cleavage was achieved using 13-nucleotide antisense oligonucleotides, including those with 1 or 3 mismatches.

[0198] In some implementations, the antisense oligonucleotide comprises the following formula: ABC, where: A contains approximately 0 to approximately 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 approximately 18 modified nucleotides; Furthermore, the full length of antisense oligonucleotides is approximately 10 to approximately 30 nucleotides.

[0199] Antisense oligonucleotides containing four or fewer DNA and / or DNA-like nucleotides in the "B" group will not recruit RNase H or guide the cleavage of the target. In these cases, the antisense oligonucleotide does not exist in a spacer aggregate mode but can act as a steric blocker.

[0200] Branched chain antisense compound This disclosure also provides branched antisense compounds comprising two or more target recognition sequences that target a portion of the EXOC2, Ku80, or Task1 nucleic acid. The branched antisense compounds of this disclosure may be, for example, branched antisense oligonucleotide compounds.

[0201] As used herein, the terms “branched antisense compound” or “branched antisense oligonucleotide” refer to two or more antisense compounds or antisense oligonucleotides linked together.

[0202] In one embodiment, the branched oligonucleotide compound comprises two or more target recognition sequences, wherein the target recognition sequences are linked to each other by one or more portions selected from adapters, spacers, and branch points. Target recognition sequences are described herein. In some embodiments, the branched oligonucleotide compound comprises 2, 3, 4, 5, 6, 7, 8, or more target recognition sequences, wherein each target recognition sequence comprises a 5' end and a 3' end, and each target recognition sequence is independently linked to an adapter, spacer, or branch point at either the 5' or 3' end. In some embodiments, each target recognition sequence is linked to an adapter, spacer, or branch point at the 5' end. In some embodiments, each target recognition sequence is linked to an adapter, spacer, or branch point at the 3' end. In another embodiment, each target recognition sequence is linked to an adapter, spacer, or branch point. In some embodiments, each of the target recognition sequences is an antisense compound and / or oligonucleotide targeting a portion of the EXOC2, Ku80, or Task1 nucleic acid.

[0203] In some embodiments, the branched oligonucleotide compounds of this disclosure have the following formula: L-(N) n Wherein N represents the target identification sequence of this disclosure; n represents an integer, such as 2, 3, 4, 5, 6, 7 or 8; and L represents a linker selected from the following: ethylene glycol chain, alkyl chain, peptide, RNA, DNA, phosphate ester, phosphonate, aminophosphate ester, ester, amide, triazole and any combination thereof.

[0204] In some embodiments, the branched oligonucleotide compounds of this disclosure have the following formula: L-(N) n 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 multivalent organic substance or a derivative thereof, and each of the one or more spacers S is independently selected from ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphate esters, phosphonates, aminophosphate esters, esters, amides, triazoles, and any combination thereof. In some embodiments, the spacers S are biolytic. For example, the spacers S may comprise portions that are readily cleaved by nucleases, proteases, pH changes, or reduction or oxidation. In such embodiments, the spacers may comprise peptides, phosphodiester-linked nucleotides, disulfide bonds, pH-sensitive linkages, or other biolytic portions. For example, the branched-chain oligonucleotide compounds of this disclosure having the formula L-(N)n have structures that are not limited in any way, such as... , , , , , , , , .

[0205] Target recognition sequence This disclosure provides an antisense oligonucleotide comprising a target recognition sequence that targets a portion of the EXOC2, Ku80, or Task1 nucleic acid. In some embodiments, the antisense oligonucleotide has a nucleobase sequence that, when written in a 5′ to 3′ orientation, contains an inverse complement of a portion of the EXOC2, Ku80, or Task1 nucleic acid. In some such embodiments, the antisense oligonucleotide has a nucleobase sequence that, when written in a 5′ to 3′ orientation, contains an inverse complement of a portion of the EXOC2, Ku80, or Task1 nucleic acid.

[0206] In some implementations, the target region is a structurally defined region of the EXOC2, Ku80, or Task1 nucleic acid. For example, the target region may encompass the 3′ untranslated region (UTR), 5′ untranslated region (UTR), exons, introns, exon / intron junctions, coding regions, translation initiation regions, translation termination regions, or other defined nucleic acid regions (e.g., open reading frames) or junctions between open reading frames and untranslated regions, and any combination thereof. The structurally defined regions of EXOC2, Ku80, and Task1 are available through accession numbers from sequence databases such as NCBI, and such information is incorporated herein by reference. In some implementations, the target region may encompass sequences from a 5′ target site within one target segment to a 3′ target site within another target segment of the same target region.

[0207] Targeting involves identifying at least one target segment that hybridizes with an antisense oligonucleotide, causing the desired effect to occur. In some embodiments, the desired effect is an increase in the level of the target nucleic acid transcript, i.e., an increase in the level of EXOC2, Ku80, or Task1 transcripts. In some 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, such as an increase in the level of EXOC2, Ku80, or Task1 proteins.

[0208] The target region may contain one or more target segments. Multiple target segments within the target region may overlap. Alternatively, they may not overlap. In some embodiments, target segments within the target region are separated by no more than about 300 nucleotides. In some embodiments, target segments within the target region are separated by about 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or by multiple nucleotides within a range determined by any two of the preceding values. In some embodiments, target segments within the target region are separated by no more than about 5 nucleotides on the target nucleic acid. In some embodiments, the target segments are continuous.

[0209] Suitable target segments may be located 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. Suitable target segments may specifically exclude a structurally defined region, such as a start or stop codon. In some embodiments, the target segment is located in intron 1 of the EXOC2, Ku80, and Task1 genes. In some embodiments, the target segment is located upstream (5') of a nucleotide repeat region in intron 1 of the EXOC2, Ku80, and Task1 genes. In some embodiments, the target segment is located downstream (3') of a nucleotide repeat region in intron 1 of the EXOC2, Ku80, and Task1 genes. In some embodiments, the target segment is located 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 has only been found once in the human genome).

[0210] Identifying suitable target regions may involve comparing the sequence of the target nucleic acid (e.g., EXOC2, Ku80, and Task1) with other sequences throughout the genome. For example, the BLAST algorithm can be used to identify regions of similarity between different nucleic acids. This comparison prevents the selection of antisense oligonucleotide sequences that can hybridize nonspecifically to sequences that are not the selected target nucleic acid (i.e., non-target or off-target sequences). Identifying suitable target regions may also 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 with similar or identical sequences across species. In some implementations, the target nucleic acid sequences of the EXOC2, Ku80, and Task1 transcripts are unique to the human genome (i.e., the target nucleic acid sequence has only been discovered once in the human genome).

[0211] When a sufficient number of antisense oligonucleotides can hydrogen-bind to the corresponding nucleobases of the target nucleic acid, the antisense oligonucleotide and the target nucleic acid (e.g., EXOC2, Ku80, and Task1 transcripts or portions thereof) are complementary to each other, so that the desired effect (e.g., increased expression of the target nucleic acid (such as EXOC2, Ku80, and Task1 transcripts or portions thereof) will occur).

[0212] Non-complementary nucleobases between antisense oligonucleotides and EXOC2, Ku80, and Task1 nucleic acids are permissible, provided that the antisense oligonucleotides maintain their ability to specifically hybridize with the target nucleic acids. Furthermore, antisense oligonucleotides can hybridize on one or more segments of EXOC2, Ku80, and Task1 nucleic acids, such that intermediate or adjacent segments do not participate in hybridization events (e.g., loop structures, mismatches, or hairpin structures).

[0213] In some embodiments, the antisense oligonucleotides provided herein or designated portions thereof are 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 and / or their target regions, target segments, or designated portions thereof, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary.

[0214] For example, an antisense oligonucleotide in which 18 of its 20 nucleotides are complementary to the target region (e.g., an equal-length portion of the EXOC2, Ku80, and Task1 transcripts) and will therefore specifically hybridize will represent 90% complementarity. In this example, the remaining non-complementary nucleotides may be clustered or interspersed with complementary nucleotides and do not need to be adjacent to each other or to complementary nucleotides. Therefore, an antisense oligonucleotide of 18 nucleotides in length with four (four) non-complementary nucleotides flanking two regions that are perfectly complementary to the target nucleic acid will have 77.8% overall complementarity to the target nucleic acid and will therefore fall within the scope of this disclosure. The percentage of complementarity between the antisense oligonucleotide and the target nucleic acid region can be routinely determined using BLAST (Basic Local Alignment Search Tool) and PowerBLAST (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656) procedures known in the art. The percentages of homology, sequence identity, or complementarity can be determined, for example, using the Gap procedure (Wisconsin Sequence Analysis Package, version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) with default settings, employing the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).

[0215] In some implementations, the antisense oligonucleotides or designated portions thereof provided herein are perfectly complementary (i.e., 100% complementary) to the target nucleic acid or designated portions thereof. For example, the antisense oligonucleotide may be perfectly complementary to the EXOC2, Ku80, or Task1 nucleic acid or its target region, target segment, or target sequence. As used herein, “perfectly complementary” or “perfectly complementary” means that each nucleobase of the antisense oligonucleotide is precisely complementary to the corresponding nucleobase of the target nucleic acid. For example, a 20-nucleobase antisense oligonucleotide may be perfectly complementary to a 400-nucleobase target sequence, provided that a corresponding 20-nucleobase portion of the target nucleic acid is perfectly complementary to the antisense oligonucleotide. Perfect complementarity may also be used with reference to designated portions of the first and / or second nucleic acids. For example, the 20-nucleobase portion of a 30-nucleobase antisense oligonucleotide may be “perfectly complementary” to a 400-nucleobase target sequence. If the target sequence has a corresponding 20-nucleobase portion in which each nucleobase is complementary to the 20-nucleobase portion of the antisense oligonucleotide, then the 20-nucleobase portion of the 30-nucleobase oligonucleotide is perfectly complementary to the target sequence. Meanwhile, the entire 30-nucleobase antisense oligonucleotide may or may not be perfectly complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense oligonucleotide are also complementary to the target sequence.

[0216] In one aspect, this disclosure provides an antisense oligonucleotide comprising a region complementary to an intron of the EXOC2, Ku80, and Task1 transcripts, wherein the antisense oligonucleotide does not comprise a region complementary to another point in the human genome (i.e., the target sequence of the antisense oligonucleotide is unique to the human genome).

[0217] In another aspect, this disclosure provides an antisense oligonucleotide containing a region complementary to intron 1 of the EXOC2, Ku80, and Task1 transcripts, wherein the antisense oligonucleotide does not contain a region complementary to another point in the human genome (i.e., the target sequence of the antisense oligonucleotide is unique to the human genome).

[0218] In some implementations, the antisense oligonucleotide contains the following sequence modification patterns. [X S ] a [ X S ] b [ S X] c ,in “a” represents an integer between 0 and 8; “b” represents an integer between 6 and 18; “c” represents an integer between 0 and 8; “s” represents the internucleotide bond between thiophosphates; "X” It consists of adenosine, guanosine, cytidine, thymine, or uracil, among which... X Contains 2'- O -(2-methoxyethyl) modification or tricyclic-DNA modification (i.e., each of adenosine, guanosine, cytidine, thymine, and uracil contains 2'- O (-(2-methoxyethyl) modification or tricyclic-DNA modification); "X" is adenosine, guanosine, cytidine, thymine, or uracil, wherein X contains a 2'-deoxy modification (i.e., each of adenosine, guanosine, cytidine, thymine, and uracil contains a 2'-deoxy modification); and The sum of a, b, and c is greater than or equal to 12.

[0219] In some implementations, the antisense oligonucleotide contains the following sequence modification patterns. 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 ,in “s” represents the internucleotide bond between thiophosphates; "X” It consists of adenosine, guanosine, cytidine, thymine, or uracil, among which... X Contains 2'- O -(2-methoxyethyl) modification or tricyclic-DNA modification; and “X” represents adenosine, guanosine, cytidine, thymine, or uracil, where X contains a 2'-deoxy modification.

[0220] In some implementations, the antisense oligonucleotide contains the following sequence modification patterns. 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 ,in “s” represents the internucleotide bond between thiophosphates; "X” It consists of adenosine, guanosine, cytidine, thymine, or uracil, among which... XContains 2'- O -(2-methoxyethyl) modified; and “X” represents adenosine, guanosine, cytidine, thymine, or uracil, where X contains a 2'-deoxy modification.

[0221] In some implementations, the antisense oligonucleotide contains the following sequence modification patterns. [X S ] d [ S X] e ,in “d” represents an integer between 0 and 40; “e” represents an integer between 0 and 40; “s” represents the internucleotide bond between thiophosphates; "X” It consists of adenosine, guanosine, cytidine, thymine, or uracil, among which... X Contains 2'- O -(2-methoxyethyl) modification or tricyclic-DNA modification; and The sum of d and e is greater than or equal to 10.

[0222] In some implementations, the antisense oligonucleotide contains the following sequence modification patterns. 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 ,in “s” represents the nucleotide bond between thiophosphates; and "X” It consists of adenosine, guanosine, cytidine, thymine, or uracil, among which... X Contains 2'- O -(2-methoxyethyl) modification or tricyclic DNA modification.

[0223] In some implementations, the antisense oligonucleotide contains the following sequence modification patterns. 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 ,in “s” represents the nucleotide bond between thiophosphates; and "X” It consists of adenosine, guanosine, cytidine, thymine, or uracil, among which... X Contains 2'- O -(2-methoxyethyl) modification.

[0224] In some implementations, each cytosine is 5-methylcytosine.

[0225] In one aspect, this disclosure provides an antisense oligonucleotide comprising the following sequence G S C S A S A S U S A S CS A S T S G S G S A S T S T S G S G S G S G ,in “s” represents the internucleotide bond between thiophosphates; "A” For containing 2'- O -(2-methoxyethyl)-modified adenosine; "G” For containing 2'- O -(2-methoxyethyl) modified guanosine; "C” For containing 2'- O -(2-methoxyethyl)-modified cytidine; "U” For containing 2'- O -(2-methoxyethyl)-modified thymine; “A” represents adenosine containing 2'-deoxy modification; “G” represents guanosine containing 2'-deoxy modification; “C” represents cytidine containing 2'-deoxymodification; and “T” represents thymine containing 2'-deoxy modification.

[0226] In one aspect, this disclosure provides an antisense oligonucleotide comprising the following sequence 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 ,in “s” represents the internucleotide bond between thiophosphates; "A” For containing 2'- O -(2-methoxyethyl)-modified adenosine; "G” For containing 2'- O -(2-methoxyethyl) modified guanosine; "C” For containing 2'- O -(2-methoxyethyl)-modified cytidine; and "U” For containing 2'- O -(2-methoxyethyl) modified thymine.

[0227] In one aspect, this disclosure provides an antisense oligonucleotide comprising the following sequence G S U S 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 “s” represents the internucleotide bond between thiophosphates; "A” For containing 2'- O -(2-methoxyethyl)-modified adenosine; "G” For containing 2'- O -(2-methoxyethyl) modified guanosine; "C” For containing 2'- O -(2-methoxyethyl)-modified cytidine; and "U” For containing 2'- O -(2-methoxyethyl) modified thymine.

[0228] In some embodiments, two or more antisense oligonucleotides are linked together by a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker degrades upon cleavage. In some embodiments, the cleavable linker is a nuclease-cleavable linker containing a phosphodiester bond. In some embodiments, the length of the nuclease-cleavable linker comprises about 2 to about 8 nucleotides. In some embodiments, the nuclease-cleavable linker comprises about 6 nucleotides. In some embodiments, the cleavable linker cleaves under reducing conditions or altered pH conditions. In some embodiments, the cleavable linker is cleaved by intracellular or endosomal nucleases. In some embodiments, the cleavable linker is cleaved by intracellular or endosomal proteases.

[0229] Conjugated antisense oligonucleotides Antisense oligonucleotides can be covalently linked to one or more moieties, ligands, sequences, or conjugates, which enhance the activity, cellular distribution, or cellular uptake of the resulting antisense oligonucleotide. These covalently linked moieties, ligands, sequences, or conjugates enhance and / or optimize pharmacokinetic parameters. These parameters include absorbance, in vivo compound concentration, extent of compound penetration, rate of compound elimination / clearance, plasma volume cleared per unit time, and other parameters.

[0230] The conjugation group may include a lysosomal ligand or sequence. In some embodiments, the lysosomal sequence is a polyG sequence. In some embodiments, the polyG sequence is a 2-30 polyG sequence. In some embodiments, the polyG sequence is a 2, 3, 4, 5, 6, 7, 8, 9, or 10 polyG sequence.

[0231] The conjugation group may include a hydrophobic portion. In one embodiment, the hydrophobic portion is selected from the group consisting of fatty acids, steroids, open-ring steroids, lipids, gangliosides and nucleoside analogs, endocannabinoids, and vitamins. In one embodiment, the steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA). In one embodiment, the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanoic acid (DCA). In one embodiment, the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, and their derivatives or metabolites. In one embodiment, the vitamin is selected from the group consisting of retinoic acid and α-tocopherol succinate.

[0232] In one embodiment, the antisense compound of this disclosure is conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand comprising a cationic group. In some 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 one exemplary embodiment, the lipophilic moiety is cholesterol. Other lipophilic moieties include cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis(hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytriphenylmethyl, or phenoxazine. A wide variety of lipid conjugates can preferentially drive oligonucleotide uptake into different tissues (Biscans et al., Nucleic Acids Res. 2019, 47, 1082-1096).

[0233] Other conjugation groups include carbohydrates, phospholipids, antibodies, peptides, biotin, phenazine, folic acid esters, phenanthridine, anthraquinones, acridine, fluorescein, rhodamine, coumarin, and dyes. In some embodiments, ligand conjugation to antisense oligonucleotides allows 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 some embodiments, the conjugate is a fibronectin type III (FN3) domain, such as centyrin protein (see, for example, Goldberg et al., Protein Eng Des Sel.2016, 29(12):563-572). The various parts, ligands, sequences or conjugates of this disclosure, as well as the means of conjugating them to antisense compounds, are described in more detail in WO2017 / 030973A1 and WO2018 / 031933A2, which are incorporated herein by reference.

[0234] Antisense oligonucleotides can also be modified to have one or more stabilizing groups, which are typically attached to one or both ends of the antisense oligonucleotide to enhance properties such as nuclease stability. The stabilizing groups include cap structures. These end modifications prevent exonuclease degradation of antisense oligonucleotides with terminal nucleic acids and aid in intracellular delivery and / or localization. The cap 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 deoxy-abase-free caps. Other 3′ and 5′-stabilizing groups that can be used to cap one or both ends of an antisense oligonucleotide to confer nuclease stability include those disclosed in WO 03 / 004602, published January 16, 2003.

[0235] In some embodiments, the antisense oligonucleotides of this disclosure comprise conjugates. In one embodiment, the antisense oligonucleotides of this disclosure comprise an antisense oligonucleotide sequence and a conjugate, wherein the conjugate is linked to the antisense oligonucleotide sequence. In some embodiments, the conjugate is selected from any of the conjugates described herein, such as hydrophobic conjugates, tissue-targeting conjugates, or conjugates designed to optimize pharmacokinetic parameters. Hydrophobic conjugates that can be conjugated to the antisense oligonucleotides of this disclosure include hexadecyloxypropyl conjugates, cholesterol conjugates, polyunsaturated fatty acid conjugates, and other hydrophobic conjugates known in the art to improve cellular uptake of the conjugated antisense oligonucleotides. In some embodiments, the conjugate may be a tissue-targeting conjugate, such as a carbohydrate conjugate or peptide conjugate, or any conjugate known in the art to target the antisense oligonucleotides of this disclosure to a specific tissue. In some embodiments, the antisense oligonucleotides of this disclosure are conjugated with a polyethylene glycol conjugate. In one embodiment, the polyethylene glycol conjugate antisense oligonucleotide optimizes the pharmacokinetic properties of the antisense oligonucleotide.

[0236] In some embodiments, this disclosure provides biolytically cleavable analogs of the antisense oligonucleotides described herein. In such cases, the biolytically cleavable analogs comprise hydrophobic conjugates that enable stronger association with cell membranes and linkers. In one embodiment, the linker is a cleavable linker that releases the antisense oligonucleotide upon cleavage (e.g., releases the antisense oligonucleotide into the endosome). In some embodiments, the antisense compound comprises a cleavable linker, wherein the cleavable linker degrades upon cleavage. In some embodiments, the linker is a nuclease-cleavable linker comprising a phosphodiester bond. In some embodiments, the length of the nuclease-cleavable linker comprising a phosphodiester bond is from about 2 to about 8 nucleotides. For example, the length of the nuclease-cleavable phosphodiester linker can be 3, 4, 5, 6, 7, 8 nucleotides or longer, such as 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 nucleotides or longer. In one embodiment, the nuclease-cleavable linker comprises about 6 nucleotides. In some embodiments, the cleavable linker cleaves after internalization into the cell. In some embodiments, the cleavable adapter cleaves within the endosome. In some embodiments, the cleavable adapter cleaves under reducing conditions. In some embodiments, the cleavable adapter cleaves under altered pH conditions, such as when the pH decreases or increases. In some embodiments, the cleavable adapter cleaves via intracellular nucleases or proteases. In some embodiments, the cleavable adapter cleaves via endosomal nucleases or proteases.

[0237] Pharmaceutical compositions and formulations This document provides pharmaceutical compositions and formulations comprising the antisense compounds described herein. For example, the antisense oligonucleotides described herein can be mixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of compounds. The pharmaceutical compositions disclosed herein are formulated to be compatible with their intended route of administration. Examples of central nervous system administration routes include intrathecal, intraventricular, or striatal administration. In some embodiments, administration may be performed using an implanted device (such as an Omaye reservoir) or an implanted intrathecal catheter. Examples of systemic administration include intravenous, subcutaneous, or intramuscular administration. The route of administration will be determined in part by the target tissue of the antisense compound. Solutions or suspensions for administration may include the following components: sterile diluents, such as water for injection, saline solution, lactated Ringer's solution, Elliott's B solution, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solutions; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, carbonates, or phosphates; and agents for adjusting tension, such as sodium chloride or dextrose. The pH may be adjusted with an acid or base (such as hydrochloric acid or sodium hydroxide). The pharmaceutical composition may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0238] Suitable pharmaceutical compositions for injectable applications include sterile aqueous solutions (wherein being water-soluble) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In all cases, the composition must be sterile and should be fluid enough to be injectable. It must be stable under manufacturing and storage conditions and must be protected against 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. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, including isotonic agents (e.g., sugars, polyols such as mannitol, sorbitol, sodium chloride) in the composition will be suitable. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0239] Sterile injectable solutions can be prepared by combining the desired amount of the active compound with one or more of the ingredients listed above (as needed) in a suitable solvent, followed by filtration sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile medium containing a base dispersion medium and any other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, some preparation methods are vacuum drying and freeze-drying, which produce a powder of the active ingredient plus any other desired components from its previously sterile filtered solution.

[0240] In some embodiments, the pharmaceutical compositions and formulations provided herein may be conveniently available in unit dosage forms and can be prepared according to conventional techniques. Such techniques may include associating the active ingredient with one or more drug carriers or one or more excipients. Generally, formulations can be prepared by homogenizing and tightly associating the active ingredient with a liquid carrier, a finely pulverized solid carrier, or both, and then (if desired) shaping the product (e.g., shaping it to a specific particle size for delivery). In one embodiment, the pharmaceutical formulation is prepared in a suitable solvent (e.g., water or physiological saline) for intrathecal, intracardiac, or striatal administration. In another embodiment, the pharmaceutical formulation is prepared in a suitable solvent (e.g., water or physiological saline) for intravenous, subcutaneous, or intramuscular administration.

[0241] The agents disclosed herein (e.g., antisense compounds targeting EXOC2, Ku80, and Task1 transcripts) can also be administered via transfection or infection using methods known in the art, including but not limited to McCaffrey et al. (2002), Nature, 418(6893), 38-9 (hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol., 20(10), 1006-10 (virus-mediated delivery); or the methods described in Putnam (1996), Am. J. Health Syst. Pharm. 53(2), 151-160, with an errata published in Am. J. Health Syst. Pharm. 53(3), 325 (1996).

[0242] The agents disclosed herein (e.g., antisense compounds targeting EXOC2, Ku80, and Task1 transcripts) can also be administered by any method suitable for administering nucleic acid agents, such as DNA vaccines. These methods include gene guns, bio-injectors, and skin patches, as well as needle-free methods such as the microparticle DNA vaccine technology disclosed in U.S. Patent No. 6,194,389 and the transdermal needle-free administration of mammalian vaccines in powder form as disclosed in U.S. Patent No. 6,168,587. Additionally, intranasal delivery is possible, as particularly as described in Hamajima et al. (1998), Clin. Immunol. Immunopathol., 88(2), 205-10. Liposomes (e.g., as described in U.S. Patent No. 6,472,375) and microencapsulation can also be used. Biodegradable, targeted microparticle delivery systems (e.g., as described in U.S. Patent No. 6,471,996) can also be used.

[0243] In one embodiment, the active agent is prepared together with a carrier that prevents the rapid elimination of the compound from the body, such as in controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials are also commercially available from Alza and Nova Pharmaceuticals. Liposome suspensions (including liposomes targeting infected cells with 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, such as those described in U.S. Patent No. 4,522,811.

[0244] The toxicity and therapeutic efficacy of such compounds can be determined using standard pharmaceutical procedures, such as those used in cell cultures or laboratory animals, to determine the LD50 (50% lethal dose) and ED50 (50% therapeutically effective dose). The dose ratio between toxicity and therapeutic effect is the therapeutic index, and it can be expressed as the ratio LD50 / ED50. Compounds exhibiting a large therapeutic index are suitable. Although compounds exhibiting toxic side effects can be used, delivery systems targeting such compounds to affected tissue sites should be carefully designed to minimize potential damage to uninfected cells and thus reduce side effects.

[0245] Data obtained from cell culture assays and animal studies can be used to determine the range of doses for human use. Doses of such compounds exhibit little or no toxicity within a circulating concentration range including the ED50. The dose can vary within this range depending on the dosage form used and the route of administration employed. For any compound used in the methods of this disclosure, a therapeutically effective dose can be initially assessed from cell culture assays. Doses can be formulated in animal models to achieve a range of circulating plasma concentrations including the EC50 (i.e., the concentration of the test compound that achieves a half-maximal response) as determined in cell cultures. This information can be used to more accurately determine the doses available in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0246] The pharmaceutical composition may be included in a container, package, or dispenser along with the instructions for use.

[0247] Antisense compounds targeting EXOC2, Ku80, and Task1 nucleic acids can be used in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. Pharmaceutically acceptable diluents include phosphate-buffered saline (PBS). PBS is suitable for use in compositions intended for parenteral delivery. Therefore, in one embodiment, the method described herein uses a pharmaceutical composition comprising antisense compounds targeting EXOC2, Ku80, and Task1 nucleic acids and a pharmaceutically acceptable diluent. In some embodiments, the pharmaceutically acceptable diluent is PBS. In some embodiments, the antisense compound is an antisense oligonucleotide.

[0248] In some implementations, the pharmaceutically acceptable diluent is designed to mimic the composition of cerebrospinal fluid. Therefore, it may contain divalent salts, such as Mg²⁺. 2+ and Ca 2+ Elliotts B solution is a suitable diluent for compositions intended to be delivered into cerebrospinal fluid. Those skilled in the art will recognize that other buffer solutions with varying concentrations of monovalent and divalent ions may also be suitable as pharmaceutically acceptable diluents.

[0249] Pharmaceutical compositions containing antisense compounds encompass any pharmaceutically acceptable salt, ester, or salt of such esters, or any other oligonucleotide capable of providing (direct or indirect) a biologically active metabolite or its residues upon administration to animals (including humans). Thus, by way of example, this disclosure also relates to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. Prodrugs may include other nucleosides cleaved by endogenous nucleases in vivo at one or both ends of the antisense compound to form the active antisense compound.

[0250] Treatment This disclosure provides a method for treating a subject with EXOC2, Ku80, or Task1-related conditions. The treatment method includes administering an effective amount of the antisense compound described herein to the subject in need. In some embodiments, the antisense compound comprises a target recognition sequence that is fully complementary to the EXOC2, Ku80, or Task1 nucleic acid (e.g., EXOC2, Ku80, or Task1 transcript) to guide RNase H to cleave the EXOC2, Ku80, or Task1 nucleic acid. In some embodiments, the antisense compound comprises a target recognition sequence that is fully complementary to the EXOC2, Ku80, or Task1 nucleic acid (e.g., EXOC2, Ku80, or Task1 transcript) to increase the expression of the EXOC2, Ku80, or Task1 nucleic acid.

[0251] Methods for treating subjects with EXOC2, Ku80, or Task1-related conditions may be used to treat any EXOC2, Ku80, or Task1-related conditions known to those skilled in the art.

[0252] In another aspect, this disclosure provides a method for treating or managing amyotrophic lateral sclerosis (ALS) in a patient, the method comprising administering to the patient a therapeutically effective amount of an oligonucleotide complementary to SOD1 as described herein.

[0253] In another aspect, this disclosure provides a method for treating or managing primary hyperlipidemia in a patient, the method comprising administering to the patient a therapeutically effective amount of dsRNA targeting PCSK9 as described herein.

[0254] In some implementation schemes, primary hyperlipidemia is defined as heterozygous familial hypercholesterolemia (HeFH).

[0255] In another aspect, this disclosure provides a method for reducing low-density lipoprotein cholesterol (LDL-C) in a patient, the method comprising administering to the patient a therapeutically effective amount of dsRNA targeting PCSK9 as described herein.

[0256] The contents of articles, patents, and patent applications mentioned or cited herein, as well as all other documents and information available electronically, are incorporated herein by reference in their entirety as if each individual publication were specifically and individually instructed to be incorporated by reference. The applicant reserves the right to actually incorporate any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents into this application.

[0257] While this disclosure has been described with reference to specific embodiments thereof, those skilled in the art will understand that various changes and alternative equivalents may be made 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 alterations may be made to the methods described herein using appropriate equivalents without departing from the scope of the embodiments disclosed herein. Furthermore, numerous modifications may be made to adapt particular circumstances, materials, composition, processes, or one or more process steps to the objectives, spirit, and scope of this disclosure. All such modifications are intended to be within the scope of the appended claims. Certain embodiments have now been described in detail and will be more clearly understood by referring to the following examples, which are included for illustrative purposes only and are not intended to be limiting.

[0258] Example The present disclosure is further illustrated by the following embodiments, which should not be construed as further limiting.

[0259] Example 1 - Materials and Methods iPSC culture Previously, from C9ORF72 The vector and its syngeneic control iPSC line were characterized (Lopez-Gonzalez et al., 2019). iPSCs were cultured (Lopez-Gonzalez et al., 2019). In short, iPSCs were maintained in 6-well plates coated with Matrigel (catalog number 354230, Corning) at a 1:100 dilution in KnockOut DMEM / F-12 (catalog number 12660012, Gibco) in mTeSR1 basal medium (mTeSR1 5X supplement, Stem Cell Technologies). The mTeSR1 medium was changed daily. iPSCs were passaged every 4–6 days. Cells were washed with DPBS and dissociated at room temperature (RT) in Accutase (catalog number SCR005, Millipore, 1:2 diluted in DPBS) for approximately 1 min. Cells were washed with DPBS and scraped from fresh mTeSR1 medium using a cell lifter. Once the desired colony size was reached, cells were seeded into matrix gel-coated 6-well plates in fresh mTeSR1 medium. Spontaneously differentiated colonies were manually removed prior to seeding and motor neuron differentiation.

[0260] Motor neuron differentiation Based on a modified protocol by Du et al. (2015), iPSCs were differentiated into spinal motor neurons (Du et al., 2015; Lopez-Gonzalez et al., 2019). Small colony-sized iPSCs were seeded in matrix gel-coated 6-well plates, with approximately 50% confluence in mTeSR1 medium. The next day, the culture medium was replaced with neural epithelial progenitor (NEP) induction medium, which consisted of KnockOut DMEM / F-12 medium (catalog number 12660012, Gibco) and neurobasal medium (catalog number 21103049, Gibco) (1:1), 0.5X N2 supplement (catalog number 17502-048, Gibco), 0.5X B27 supplement (catalog number 17504044, Gibco), 0.1 mM ascorbic acid (catalog number A4403, Sigma), 1X Glutamax (catalog number 35050061, Thermo Fisher Scientific), supplemented with 3 μM CHIR99021 (catalog number 72054, Stem Cell Technologies), 2 μM DMH1 (catalog number 4126, TocrisBioscience), and 2 μM SB431542. (Catalogue No. 04-0010-10, Stemgent). NEP culture medium was changed every other day for 6 days. NEPs were dissociated with a 1:2 dilution of Accutase and seeded 1:3 into matrix gel-coated 6-well plates. NEPs were cultured in motor neuron progenitor (MNP) induction medium supplemented with 1 μM CHIR99021, 2 μM DMH1, 2 μM SB431542, 0.1 μM retinoic acid (Catalogue No. R2625, Sigma), and 0.5 μM purmorphamine (Catalogue No. 540220, Calbiochem). MNP culture medium was changed every other day for 6 days. MNPs were dissociated with a 1:2 dilution of Accutase and cultured in suspension in 60 mm low-attachment plates in motor neuron differentiation medium supplemented with 0.5 μM retinoic acid and 0.1 μM purmorphamine for 6 days. The culture medium was changed every other day for 6 days.Finally, neurospheres were dissociated into single cells using Accutase (10 min, 37°C), passed through a 40 μm filter, and introduced into motor neuron culture medium supplemented with 0.5 μM retinoic acid, 0.1 μM puromorphamine, 0.1 μM compound E (catalog number 73954, Stem Cell Technologies), 10 ng / ml BDNF (catalog number 248-BDB, R&D Systems), 10 ng / ml GDNF (catalog number PHC7041, Thermo Fisher Scientific), and 1 μg / ml laminin (catalog number L2020, Sigma). Cells were cultured at 5 x 10⁻⁶ cells / mL. 5 Neurons were seeded at a density of 10 cells / well in matrix gel-coated 12-well plates in motor neuron medium. After two weeks, neurons were maintained in motor neuron medium without retinoic acid and pomofoamine. Half of the medium was replaced weekly for three weeks.

[0261] Cell culture 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 Durbecco's Modified Eagle's 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 cultured at 2 x 10⁻⁶ cells per cell line. 5 Cells were seeded at a density of 10 cells / well in 12-well plates for processing.

[0262] ASO processing Three-week-old iPSC-derived neurons or cell lines were treated for two days with a specified concentration of ASO. 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.

[0263] Non-targeted control (NTC) EXOC2-349, EXOC2-3933, Ku80-624, Ku80-1425, Ku80-2802, Task1-692, Task1-5968 The ASO was designed by IDT Custom Design Service. All ASOs listed in Table 1 were purchased from IDT.

[0264] Bafifimimycin A1 treatment One day after inoculation, HEK293T cells were treated with 50 nM bafimib A1 (BafA1) or DMSO for 24 hours. The next day, the cells were treated with ASO and 10 nM BafA1. Cells were harvested two days after ASO treatment for RNA extraction.

[0265] RNA extraction and real-time quantitative PCR Total RNA was extracted from iPSC-MN using the RNeasy Mini kit (catalog number 74106, Qiagen). 1 μg of RNA was reverse transcribed into cDNA using the TaqMan reverse transcription kit (catalog number N8080234, Thermo Fisher Scientific) with random hexamer. Real-time quantitative PCR was performed using the Applied Biosystems Quant Studio 3 system with SYBR Select premixed buffer (catalog number 4472918, Thermo Fisher Scientific). Ct values ​​for each sample were calculated. Parental ring white Standardization. Use 2 -ΔΔCt The relative mRNA level of each gene was calculated using a method. Table 2 lists the qRT-PCR primers.

[0266] Example 2 - Spacer polymers with and without polyG, polyA, or polyC ligands, as measured by qRT-PCT analysis EXOC2 mRNA levels The relative strengths of spacer polymers with and without poly-G, poly-A, or poly-C ligands, as measured by qRT-PCT analysis, were evaluated. EXOC2 mRNA levels ( Figure 3 ).

[0267] Relative assays were performed on HEK cells treated for two days with 5 μM of untargeted control (NTC) or the EXOC2-targeting spacer polymers ASO 3933, LysoASO 3933-G5, or ASO 3933-A5 or 3933-C5 with other sequences. EXOC2 qRT-PCR analysis of mRNA levels.

[0268] The ASO sequence with the poly-G ligand was found to be specific and provided additional knockdown compared to ASO without the poly-G ligand. ASO 3933 was found to be significantly reduced compared to NTC ASO. EXOC2mRNA levels were found to significantly increase the knockdown efficiency of the spacer aggregate ASO 3933 by adding the Lyso sequence (GGGGG) (3933-G5). This enhanced knockdown efficiency was specific to the Lyso sequence, as adding (AAAAA) or (CCCCC) to 3933 did not significantly alter the relative mRNA levels compared to 3933. EXOC2 mRNA levels.

[0269] Example 3 - Spacer polymers with and without poly-G ligands of different lengths, as measured by qRT-PCT analysis EXOC2 mRNA levels Spacer polymers with and without poly-G ligands of different lengths were evaluated, as measured by qRT-PCT analysis. EXOC2 mRNA levels ( Figure 4 ).

[0270] Using 5 uM NTC, targeted EXOC2 HEK cells were treated for two days with spacer polymer ASO 3933 and LysoASO with different G lengths (3933-G4, 3933-G5, 3933-G10, and 3933-G12) for relative EXOC2 qRT-PCR analysis of mRNA levels.

[0271] The asoylase sequence with poly-G ligands was found to have a range of 5-10 G. ASO3933 was found to be significantly lower than NTC ASO. EXOC2 mRNA levels were found to be significantly increased by ASO 3933-G5 and 3933-G10, leading to a significant increase in the knockdown efficiency of the spacer aggregate ASO 3933. ASO 3933-G4 and 3933-G12 did not decrease mRNA levels. EXOC2 mRNA levels.

[0272] Example 4 - EXOC2 349, as measured by qRT-PCT analysis EXOC2 mRNA levels Evaluation EXOC2 349. As measured by qRT-PCT analysis, relative EXOC2 mRNA levels ( Figure 5 ).

[0273] Using 5 uM NTC, targeted EXOC2 iPSC-derived neurons were treated for two days with spacer polymers ASO 349, LysoASO 349-G10, 349-G7 PS, and other unmodified oligonucleotides (349 unmodified, 349 unmodified G10, 349 unmodified with G7 side-jointed) for relative EXOC2qRT-PCR analysis of mRNA levels.

[0274] ASOs with poly-G ligands were found to restore the function of inactive ASOs in iPSC-derived neurons. Spacing polymeric ASO 349 was found to have no relative change compared to NTC ASOs. EXOC2 mRNA levels were found to be significantly lower in ASO349-G10 compared to NTC or ASO 349. EXOC2 mRNA levels. Compared with NTC ASO, ASO 349-G7 PS did not change the relative EXOC2 mRNA level, indicating that complete or partial PS modification can prevent the Lamp2C receptor from recognizing the Lyso sequence.

[0275] Example 5 - EXOC2 349, as measured by qRT-PCT analysis EXOC2 mRNA levels Evaluation EXOC2 3933, as measured by qRT-PCT analysis, relative EXOC2 mRNA levels ( Figure 6 ).

[0276] Using 5 uM NTC, targeted EXOC2 Wild-type (WT) and LAMP2 knockout (KO) HeLa cells were treated for two days with spacer polymer ASO 3933 and LysoASO 3933-G5 for relative... EXOC2 qRT-PCR analysis of mRNA levels. Compared with NTCASO, spacer polymer ASO 3933 significantly reduced relative mRNA levels in WT and LAMP2 KO cells. EXOC2 mRNA levels.

[0277] The effects of LysoASO were found to be mediated by the LAMP2 receptor. LysoASO 3933-G5 was found to further increase the knockdown efficiency of ASO3933. In WT cells, compared to the ASO 3933 group, the LysoASO 3933-G5 group showed significantly higher knockdown efficiency. EXOC2 mRNA levels were significantly reduced. However, this effect was not observed in LAMP2 KO cells, and LysoASO3933-G5 remained unchanged compared to NTC or ASO 3933. EXOC2 mRNA levels.

[0278] Example 6 - Lysosomal inhibition and non-inhibition conditions EXOC2 3933, as measured by qRT-PCT analysis EXOC2 mRNA levels The effects of lysosomal inhibition and non-inhibition were evaluated. EXOC23933, as measured by qRT-PCT analysis, relative EXOC2 mRNA levels ( Figure 7 and Figure 8 ).

[0279] Using 5 uM NTC, targeted EXOC2 qRT-PCR analysis of relative EXOC2 mRNA levels was performed in HEK cells treated for two days with spacer polymers ASO 3933 and LysoASO 3933-G5. Prior to ASO treatment, HEK cells were treated for 24 hours with DMSO or a 50 nM bafimycin A1 (Baf A1) lysosomal inhibitor.

[0280] LysoASO was found to have effects mediated via functional lysosomes. Compared to NTC ASO, spacer polymer ASO3933 significantly reduced the relative levels of DMSO and Baf A1 in cells treated with these compounds. EXOC2 mRNA levels. LysoASO 3933-G5 was found to further increase the knockdown efficiency of ASO 3933. In DMSO-treated cells, EXOC2 mRNA levels were significantly lower in the LysoASO3933-G5 group compared to the ASO 3933 group. However, this effect was eliminated in Baf A1-treated cells; LysoASO 3933-G5 did not further reduce levels compared to ASO3933. EXOC2 mRNA levels.

[0281] Example 7 - Spatially blocked and unblocked EXOC2 3933 as measured by qRT-PCT analysis EXOC2 mRNA levels The spatial blocking and unblocking were assessed. EXOC2 3933, as measured by qRT-PCT analysis, relative EXOC2 mRNA levels ( Figure 9 and Figure 10 ).

[0282] Using 5 uM NTC, targeted EXOC2 Spatial blocking ASO 3933 and LysoASO 3933-G5 spatial blocking ASO treatment for two days were used to perform relative... EXOC2 qRT-PCR analysis of mRNA levels.

[0283] LysoASO was found to target RNA for degradation via a non-RNase H-dependent pathway. Unsurprisingly, compared to NTC, steric blocking of ASO 3933 did not alter the relative... EXOC2mRNA levels were found to be significantly reduced by LysoASO steric occlusion of ASO 3933-G5 compared to NTC. EXOC2 The level of mRNA indicates its involvement in the degradation pathway of alternative RNase H.

[0284] Example 8 - Ku80 624 and 2802 as measured by qRT-PCT analysis Ku80 mRNA levels The relative strengths of Ku80 624 and 2802, as measured by qRT-PCT analysis, were evaluated. Ku80 mRNA levels ( Figure 11 ).

[0285] In (A) using 1 uM NTC, targeted Ku80 In iPSC-derived neurons treated with spacer polymers ASO 624 and 1425, LysoASO 624-G10 and 1425-G10, (B) HeLa cells treated with 1 uM NTC, Ku80-targeting spacer polymers ASO 2802 and LysoASO 2802-G10 (treated for two days) were compared. Ku80 qRT-PCR analysis of mRNA levels.

[0286] LysoASO was found to be effective in inducing Ku80 mRNA degradation was observed. Compared to NTC, spacer aggregates ASO 624 and 1424 were found to be significantly reduced. Ku80 mRNA levels were found to be further reduced by LysoASO 624-G10 and 1425-G10 compared to the spacer polymers ASO 624 and 1424, respectively. Ku80 mRNA levels were found to be unchanged compared to NTC. Ku80 mRNA levels were found to be significantly lower in LysoASO 2802-G10 compared to NTC and ASO 2802. Ku80 mRNA levels.

[0287] Example 9 - TASK1 622 and 5968 as measured by qRT-PCT analysis TASK1 mRNA levels Evaluation TASK1 622 and 5968, as measured by qRT-PCT analysis, are relative values. TASK1 mRNA levels ( Figure 12 ).

[0288] Using 5 uM NTC, targeted Task 1The spacer polymers ASO 692 and 5968, LysoASO 692-G10 and 5968-G10 were used to treat iPSC-derived neurons for two days to perform relative... Task 1 qRT-PCR analysis of mRNA levels.

[0289] LysoASO was found to be effective in inducing Task 1 mRNA was degraded. It was found that ASO 692 remained unchanged compared to NTC. Task 1 mRNA levels were found to be significantly lower in ASO 5968 compared to NTC. Task 1 mRNA levels were found to be significantly lower in LysoASO 692-G10 and 5968-G10 compared to ASO692 and 5968, respectively. Task 1 The level of mRNA and the activation of the inactive ASO 692.

[0290] Example 10 - Enhancing the silencing of approved oligonucleotide therapeutics with polyG ligands The advantage of the polyG ligand described in this article is its ability to enhance the broad efficacy of silencing in any therapeutic oligonucleotide, including approved oligonucleotide therapeutics that have been selected for robust silencing efficacy. To demonstrate this efficacy, two approved oligonucleotide therapeutics, anti-SOD1 ASO thovsen (QALSODY®) and anti-PCSK9 siRNA incoxlane (LEQVIO®), were tested in the presence and absence of the polyG ligand.

[0291] Example 10 - Materials and Methods Cell culture SH-SY5Y cells were maintained in DMEM / F-12 (Dürbeck Modified Eagle Medium / Nutrient Mixture F-12) supplemented with 10% FBS (fetal bovine serum) and 1% penicillin / streptomycin. A549 cells were maintained in F-12 medium supplemented with 10% FBS and 1% Pen-strep. Cells were cultured at 2 x 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 10 cells / well in 24-well plates for processing.

[0292] ASO processing Using liposome RNAiMAX, SH-SY5Y cells were transfected with 25 or 100 nM ASO as listed in Table 3. Cells were harvested 24 hours after ASO treatment for RNA extraction and RT-qPCR analysis.

[0293] siRNA treatment The sense siRNA was hybridized to the antisense strand or Lamp-antisense strand. A549 cells were transfected with 50 nM siRNAs listed in Table 4 using liposome RNAiMAX. Cells were harvested 48 hours after siRNA treatment for RNA extraction and RT-qPCR analysis.

[0294] RNA extraction and real-time quantitative PCR Total RNA was extracted using acidic guanidinol-chloroform thiocyanate (QIAzol reagent, Qiagen) following the manufacturer's protocol. 1 μg of RNA was reverse transcribed into cDNA using random hexamer and ProtoScript II reverse transcriptase (New England Biolabs).

[0295] Real-time quantitative PCR (RT-qPCR) was performed using a Bio-rad CFX96 real-time PCR instrument. The Ct values ​​for each sample were then calculated. GAPDH Standardization. Use 2 -ΔΔCt The relative mRNA level of each gene was calculated using a method. Table 5 lists the qRT-PCR primers.

[0296] Example 10 - Results ASO toffsen 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). Figure 13 As shown, compared to the non-target control, the silencing efficacy of toffsen was significantly improved with the inclusion of a 10G ligand, based on relative SOD1 expression. Alternative polyG ligands were also tested using toffsen (a toffsen-Lamp derivative). These alternative polyG ligands do not contain consecutive G nucleotides, but instead have an intermediate non-G nucleotide (A nucleotide) after every three G nucleotides. Figure 15 As shown, the alternative polyG ligand is just as effective as the continuous G ligand 10G in enhancing SOD1 inhibition.

[0297] Incoxlane siRNA was tested with and without the poly-G ligand. In this case, the 5G ligand was used at the 3' end of the antisense strand. A549 cells were treated with 50 nM incoxlane or incoxlane-Lamp for 48 hours. Figure 14 As shown, compared with the non-target control, the silencing efficacy of inxladesh was also significantly improved in the presence of poly-G ligand (5G ligand) based on relative PCSK9 expression.

[0298] Importantly, both diagrams demonstrate the enhanced silencing efficacy of polyG ligands in two different types of therapeutic oligonucleotides (ASO and siRNA). Furthermore, polyG ligands enhance silencing in therapeutic oligonucleotides containing a variety of chemical modifications.

[0299] This work confirms that polyG ligands are widely applicable to any oligonucleotide type, regardless of the use of the base oligonucleotide sequence or chemical modifications.

[0300] sequence Table 1 - ASO.

[0301] Note: (-) indicates the phosphodiester backbone, (*) indicates the thiophosphate backbone. Bold (N) indicates an unmodified nucleotide, while bold italic (N) indicates an unmodified nucleotide. N () indicates nucleotides modified with 2′-O-methoxy-ethyl (2′MOE) sugar.

[0302] Table 2 - qRT-PCR primers.

[0303] Table 3. ASO sequence. (#) indicates a phosphate thioester bond. (eN) indicates a 2' MOE, (dN) indicates a standard 2' deoxyribonucleoside, and (d5N) indicates a 5-methyl-dN (e.g., 5-methylcytosine).

[0304]

[0305] Table 4. siRNA sequences. (#) indicates a phosphate thioester bond. (mN) indicates 2'-OMe, (fN) indicates 2'-fluoronucleotide modification, and V indicates 5'-vinyl phosphate.

[0306]

[0307] Table 5. qRT-PCR primers.

[0308] References

[0309]

[0310]

Claims

1. An oligonucleotide comprising a 5' end, a 3' end, and complementarity with a target polynucleotide, wherein the oligonucleotide comprises a polyG sequence attached to the 5' end and / or the 3' end of the oligonucleotide, and wherein the polyG sequence lacks complementarity with the target polynucleotide.

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

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

5. The oligonucleotide of any one of claims 1-3, wherein the polyG sequence comprises or consists of 6 G nucleotides.

6. The oligonucleotide of any one of claims 1-3, wherein the polyG sequence comprises or consists of 7 G nucleotides.

7. The oligonucleotide of any one of claims 1-3, wherein the polyG sequence comprises or consists of 8 G nucleotides.

8. The oligonucleotide of any one of claims 1-3, wherein the polyG sequence comprises or consists of 9 G nucleotides.

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

10. The oligonucleotide of any of the preceding claims, wherein the polyG sequence comprises continuous, discontinuous, or combinations thereof G nucleotides.

11. The oligonucleotide of any one of claims 1-10, wherein the polyG sequence comprises (dG)(dG)(dG)(dG)(dG) or is composed of therewith.

12. The oligonucleotide of any one of claims 1-10, wherein the polyG sequence comprises (dG)(dG)(dG)(dG)(dG)(dG)(dG) or is composed of therewith.

13. The oligonucleotide of any one of claims 1-10, wherein the polyG sequence comprises (dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG) or is composed of therewith.

14. The oligonucleotide of any one of claims 1-10, wherein the polyG sequence comprises (dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG) or is composed of therewith.

15. The oligonucleotide of any one of claims 1-10, wherein the polyG sequence comprises (dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG) or is composed of therewith.

16. The oligonucleotide of any one of claims 1-10, wherein the polyG sequence comprises (dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG) or is composed of therewith.

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

18. The oligonucleotide of any one of claims 1-10, wherein the polyG sequence comprises (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dN)(dG)(dG) or is composed of therewith, wherein each dN individually corresponds to a deoxyribonucleotide of dA, dT or dC.

19. The oligonucleotide of any one of claims 1-10, wherein the polyG sequence comprises (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dN)(dG)(dG) or is composed of therewith, wherein each dN individually corresponds to a deoxyribonucleotide of dA, dT or dC.

20. The oligonucleotide as claimed in any of the preceding claims, wherein the length of the oligonucleotide is from about 10 nucleotides to about 35 nucleotides.

21. The oligonucleotide as claimed in any of the preceding claims, wherein the length of the oligonucleotide is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

22. The oligonucleotide as claimed in any of the preceding claims, wherein the oligonucleotide and / or the polyG sequence comprises one or more modified nucleotides.

23. The oligonucleotide of claim 22, wherein the one or more modified nucleotides each independently comprises a ribose group, a phosphate group, a nucleobase, or a combination thereof.

24. The oligonucleotide of claim 23, wherein each modification of the ribose group comprises 2'-O-methyl, 2'-fluorine, 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 of claim 24, wherein the restricted nucleotide comprises locked nucleic acid (LNA), ethyl-restricted nucleotide, 2'-(S)-restricted ethyl (S-cEt) nucleotide, restricted MOE, 2'-O,4'-C-aminomethylene bridging nucleic acid (2',4'-BNANC), α-L-locked nucleic acid, tricyclic DNA, or combinations thereof.

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

27. The oligonucleotide of 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 the 3' end of the oligonucleotide comprise 2'-O-(2-methoxyethyl) (MOE) modification.

28. The oligonucleotide of claim 24, wherein each nucleotide of the oligonucleotide and / or the polyG sequence comprises a 2'-O-(2-methoxyethyl) (MOE) modification.

29. The oligonucleotide of claim 23, wherein the modification of the ribose group comprises a tricyclic DNA modification.

30. The oligonucleotide of claim 23, wherein each nucleotide of the oligonucleotide and / or the polyG sequence comprises a tricyclic DNA modification.

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

32. The oligonucleotide of claim 23, wherein each modification of the phosphate group comprises a thiophosphate, a phosphonoacetate (PACE), a thiophosphonoacetate (thioPACE), an amide, a triazole, a phosphonate, a triphosphate, or a combination thereof.

33. The oligonucleotide of claim 23, wherein the modification of the phosphate group is a thiophosphate ester.

34. The oligonucleotide of claim 23, wherein each nucleotide of the oligonucleotide and / or the polyG sequence comprises a phosphate thioester.

35. The oligonucleotide as claimed in any of the preceding claims, wherein the oligonucleotide and / or the polyG sequence comprises at least one phosphodiester nucleotide inter-bond.

36. The oligonucleotide as claimed in any of the preceding claims, wherein each nucleotide bond in the oligonucleotide and / or the polyG sequence is a phosphodiester nucleotide bond.

37. The oligonucleotide of claim 23, wherein each modification of the nucleobase comprises 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.

38. The oligonucleotide of claim 23, wherein the modification of the nucleobase group comprises 5-methylcytosine modification.

39. The oligonucleotide as claimed in any of the preceding claims, wherein the oligonucleotide comprises a mixture of modified nucleotides.

40. The oligonucleotide as claimed in any of the preceding claims, wherein the functional portion is attached to the 5' end or the 3' end of the oligonucleotide.

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

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

43. The oligonucleotide as claimed in any of the preceding claims, wherein the oligonucleotide comprises the following formula: ABC, where: A contains approximately 0 to approximately 8 modified nucleotides; B contains approximately 6 to approximately 18 deoxyribonucleic acid (DNA) nucleotides and / or DNA-like nucleotides; and C contains approximately 0 to approximately 8 modified nucleotides; Furthermore, the full length of the antisense oligonucleotides is approximately 10 to approximately 30 nucleotides.

44. The oligonucleotide of 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 of claim 43 or 44, wherein A comprises about 5 modified nucleotides, B comprises about 10 DNA nucleotides and / or DNA-like nucleotides, and C comprises about 5 modified nucleotides.

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

47. The oligonucleotide of claim 43 or 44, wherein 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.

48. The oligonucleotide as claimed in any of the preceding claims, wherein the oligonucleotide comprises a nucleic acid sequence having at least 90% sequence identity with any of the nucleic acid sequences in Table 1.

49. The oligonucleotide as claimed in any of the preceding claims, wherein the oligonucleotide comprises the following sequence 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 X S X S X S X S X S X S X , where s represents the internucleotide bond between thiophosphates; X It contains adenosine, guanosine, cytidine, thymine, or uracil, among which X Contains 2'- O -(2-methoxyethyl) modified; and X contains adenosine, guanosine, cytidine, thymine, or uracil, wherein X contains a 2'-deoxy modification.

50. The oligonucleotide as claimed in any of the preceding claims, wherein the oligonucleotide comprises the following sequence 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 X S X S X S X S X ,in 's' represents the nucleotide internucleotide bond of a phosphate thioester; and X It contains adenosine, guanosine, cytidine, thymine, or uracil, among which X Contains 2'- O -(2-methoxyethyl) modification.

51. The oligonucleotide of any of the preceding claims, wherein the target is mammalian or viral mRNA, optionally wherein the target is an intronic or exon region of the mRNA.

52. The oligonucleotide of any of the preceding claims, wherein the target is selected from the group consisting of: EXOC2 Gene, Ku80 Genes and Task 1 Gene.

53. The oligonucleotide of claim 52, wherein the oligonucleotide comprises the EXOC2 Genes, the aforementioned Ku80 Gene or the stated Task 1 Gene expression is suppressed by at least approximately 50%.

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

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

56. The oligonucleotide of any one of claims 1-51, wherein the oligonucleotide comprises the sequence (eC)#(eA)(eG)#(eG)(eA)#(dT)#(dA)#(d5C)#(dA)#(dT)#(dT)#(d5C)#(dT)#(dA)#(eC)(eA)#(eG)(eC)#(eU)(dG) x , where (#) indicates a thiophosphate bond, (e) indicates a 2'MOE modification, (d) indicates a deoxyribonucleotide, (d5C) indicates 5-methylcytosine, and x indicates an integer between 5 and 10.

57. The oligonucleotide of any one of claims 1-51, wherein 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)(dGdGdGdN) x , where (#) indicates a thiophosphate bond, (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 between 2 and 10.

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

59. The oligonucleotide of any one of claims 1-51, wherein the oligonucleotide is selected from the group consisting of: antisense oligonucleotides (ASO), spacer polymers, siRNA, miRNA, shRNA, CRISPR guides, DNA, antisense hybrids, miRNA inhibitors, splice-conversion oligonucleotides (SSO), diaminophosphate morpholino oligomers (PMO), and peptide nucleic acids (PNA).

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

61. The dsRNA of claim 60, wherein the dsRNA comprises: An antisense strand complementary to the target, wherein the length of the antisense strand is optionally about 10-35 nucleotides; A sense strand complementary to at least a portion of the antisense strand, optionally wherein the length of the sense strand is about 10-35 nucleotides; and A polyG sequence connected to the 5' end and / or 3' end of the antisense strand and / or the sense strand.

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

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

64. The dsRNA according to any one of claims 60-63, wherein the dsRNA comprises an antisense strand of V(mA)#(fC)#(mA)(fA)(fA)(fA)(mG)(fC)(mA)(fA)(mA)(mA)(mC)(fA)(mG)(fG)(mU)(fC)(mU)(mA)(mG)#(mA)#(mA)(polyG), wherein (#) indicates a thiophosphate bond, (mN) indicates 2'-OMe modification, (fN) indicates 2'-fluorine modification, V indicates 5'-vinyl phosphate, and (polyG) indicates 2-30 G nucleotides.

65. The dsRNA according to any one of claims 60-63, wherein the dsRNA comprises V(mA)#(fC)#(mA)(fA)(fA)(fA)(mG)(fC)(mA)(fA)(mA)(mA)(mA)(mC)(fA)(mG)(fG)(mU)(fC)(mU)(mA)(mG)#(mA)#(mA)(dG) x The antisense chain, where (#) indicates a thiophosphate bond, (mN) indicates 2'-OMe modification, (fN) indicates 2'-fluorine modification, V indicates 5'-vinylphosphine, and x indicates an integer between 5 and 10.

66. A pharmaceutical composition for inhibiting gene expression in an organism, said pharmaceutical composition comprising an oligonucleotide as described in any one of claims 1-59 or dsRNA as described in any one of claims 60-65 and a pharmaceutically acceptable vector.

67. The pharmaceutical composition of claim 66, wherein the gene is selected from the group consisting of: EXOC2 Gene, Ku80 Genes and Task 1 Gene.

68. The pharmaceutical composition of claim 67, wherein the oligonucleotide or the dsRNA delivers the... EXOC2 Genes, the aforementioned Ku80 Gene or the stated Task 1 Gene expression is suppressed by at least approximately 50%.

69. The pharmaceutical composition of claim 68, wherein the oligonucleotide or the dsRNA delivers the... EXOC2 Genes, the aforementioned Ku80 Gene or the stated Task 1 Gene expression is suppressed by at least approximately 80%.

70. A vector comprising a regulatory sequence operatively linked to a nucleotide sequence encoding an oligonucleotide as described in any one of claims 1-59 or a dsRNA as described in any one of claims 60-65.

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

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

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

74. The vector according to any one of claims 70-73, wherein the gene is selected from the group consisting of: EXOC2 Gene, Ku80 Genes and Task 1 Gene.

75. A recombinant adeno-associated virus (rAAV) comprising a vector as described in any one of claims 70-74 and an AAV capsid.

76. A cell comprising the vector as described in any one of claims 70-74 or the rAAV as described in claim 75.

77. A method for suppressing gene expression in cells, the method comprising: (a) Introducing into the cells an oligonucleotide as described in any one of claims 1-59, dsRNA as described in any one of claims 60-65, a vector as described in any one of claims 70-74, or rAAV as described in claim 75; and (b) The cells produced in step (a) are maintained for a time sufficient to allow the mRNA transcript of the gene to degrade, thereby inhibiting the expression of the gene in the cells.

78. The method of claim 77, wherein the polyG sequence guides the degradation from the lysosome.

79. The method of claim 78, wherein the gene is selected from the group consisting of: EXOC2 Gene, Ku80 Genes and Task 1 Gene.

80. The method of claim 79, wherein the oligonucleotide, the dsRNA, the vector, or the rAAV delivers the... EXOC2 Genes, the aforementioned Ku80 Gene or the stated Task 1 Gene expression is suppressed by at least approximately 50%.

81. The method of claim 80, wherein the oligonucleotide, the dsRNA, the vector, or the rAAV delivers the... EXOC2 Genes, the aforementioned Ku80 Gene or the stated Task 1 Gene expression is suppressed by at least approximately 80%.

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

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

84. The method of claim 83, wherein the gene is selected from the group consisting of: EXOC2 Gene, Ku80 Genes and Task 1 Gene.

85. The method of claim 84, wherein the oligonucleotide, the dsRNA, the vector, or the rAAV delivers the... EXOC2 Genes, the aforementioned Ku80 Gene or the stated Task 1 Gene expression is suppressed by at least approximately 50%.

86. The method of claim 85, wherein the oligonucleotide, the dsRNA, the vector, or the rAAV delivers the... EXOC2 Genes, the aforementioned Ku80 Gene or the stated Task 1 Gene expression is suppressed by at least approximately 80%.

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

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

89. The method of claim 88, wherein the target is selected from the group consisting of: EXOC2 Gene, Ku80 Genes and Task 1 Gene.

90. The method of claim 89, wherein the oligonucleotide comprises EXOC2 Genes, the aforementioned Ku80 Gene or the stated Task 1 Gene expression is suppressed by at least approximately 50%.

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

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

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

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

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