DEGRADATION OF RNA BY THE LYSOSOMAL PATHWAY

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML file, created on Dec. 10, 2024, is named 755176_UM9-307_ST26.xml and is 137,916 bytes in size.

BACKGROUND

RNA knockdown-based approaches have emerged as powerful tools to manipulate gene expression and elucidate gene functions (Fire et al., 1998; Hannon, 2002). Utilizing small RNA molecules, such as small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), these methods offer the potential to selectively silence gene expression with high precision and specificity (Elbashir et al., 2001; Brummelkamp et al., 2002). Despite their promise, these approaches have faced challenges, particularly in terms of efficacy and target specificity (Jackson & Linsley, 2010; Boudreau et al., 2011).

One of the critical limitations encountered in RNA knockdown methodologies is related to the intracellular fate of the RNA molecules. Cellular uptake and subsequent processing are intricate processes that can profoundly influence the knockdown efficiency (Juliano, 2016). Moreover, suboptimal endosomal escape and premature degradation in the lysosomal compartments have been identified as major obstacles that hamper the effectiveness of these approaches (Gilleron et al., 2013; Daka et al., 2020).

Accordingly, there exists a need for more efficient RNA knockdown methodologies.

SUMMARY

The present disclosure provides oligonucleotides, methods, and compositions for degrading RNA via the lysosomal pathway.

In one aspect, the disclosure provides an oligonucleotide comprising a 5′ end, a 3′ end, and complementarity to a target polynucleotide, wherein the oligonucleotide comprises a poly-G sequence linked to the 5′ end and/or 3′ end of the oligonucleotide, and wherein the poly-G sequence lacks complementarity to the target polynucleotide.

In some embodiments, the poly-G sequence comprises 2-30 G nucleotides.

In some embodiments, the poly-G sequence is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 G nucleotides (SEQ ID NO: 57). 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.

In some embodiments, the poly-G sequence is linked to the 3′ end of an oligonucleotide of the disclosure. In other embodiments, the poly-G sequence is linked to the 5′ end of an oligonucleotide of the disclosure. In other embodiments, the poly-G sequence is linked to the 5′ end and the 3′ end of an oligonucleotide of the disclosure.

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

In some embodiments, the poly-G sequence binds to a protein on the surface of a lysosome. In some embodiments, the poly-G sequence binds to a lysosome-associated membrane glycoprotein (LAMP). In some embodiments, the LAMP is LAMP2C. In some embodiments, the poly-G sequence comprises G nucleotides that are consecutive. In some embodiments, the poly-G sequence comprises or consists of 5-15 consecutive G nucleotides. In some embodiments, the poly-G sequence comprises 1 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 1 or more non-G nucleotides (e.g., A, T, U, or C) between the two or more poly-G sequences.

In some embodiments, the poly-G sequence comprises G nucleotides that are consecutive, not consecutive, or a combination thereof.

In some embodiments, the poly-G sequence comprises or consists of (dG)(dG)(dG)(dG)(dG). In some embodiments, the poly-G sequence comprises or consists of (dG)(dG)(dG)(dG)(dG)(dG). In some embodiments, the poly-G sequence comprises or consists of (dG)(dG)(dG)(dG)(dG)(dG)(dG). In some embodiments, the poly-G sequence comprises or consists of (dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG). In some embodiments, the poly-G sequence comprises or consists of (dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG). In some embodiments, the poly-G sequence comprises or consists of (dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG)(dG)(SEQ ID NO: 56). In some embodiments, the poly-G sequence comprises or consists of (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dN)(dG), wherein each dN individually corresponds to a deoxyribonucleotide of dA, dT, or dC. In some embodiments, the poly-G sequence comprises or consists of (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dN)(dG)(dG)(SEQ ID NO: 58), wherein each dN individually corresponds to a deoxyribonucleotide of dA, dT, or dC. In some embodiments, the poly-G sequence comprises or consists of (dG)(dG)(dG)(dN)(dG)(dG)(dG)(dN)(dG)(dG) (SEQ ID NO: 58), wherein each dN individually corresponds to a deoxyribonucleotide of dA, dT, or dC.

In some embodiments, the oligonucleotide is about 10 nucleotides to about 35 nucleotides in length.

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

In some embodiments, the oligonucleotide and/or poly-G sequence comprise one or more modified nucleotides.

In some embodiments, the one or more modified nucleotides each independently comprise a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof.

In some embodiments, each modification of the ribose group comprises 2′-O-methyl, 2′-fluoro, 2′-deoxy, 2′-O-(2-methoxyethyl)(MOE), 2′-O-alkyl, 2′-O-alkoxy, 2′-O-alkylamino, 2′-NH2, a constrained nucleotide, or a combination thereof.

In some embodiments, the constrained nucleotide comprises a locked nucleic acid (LNA), an ethyl-constrained nucleotide, a 2′-(S)-constrained ethyl(S-cEt) nucleotide, a constrained MOE, a 2′-0, 4′-C-aminomethylene bridged nucleic acid (2′,4′-BNANC), an alpha-L-locked nucleic acid, a tricyclo-DNA, or a combination thereof.

In some embodiments, the modification of the ribose group comprises a 2′-O-(2-methoxyethyl)(MOE) modification.

In some embodiments, the nucleotides at the position 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10 from the 5′ end and/or 3′ end of the oligonucleotide comprise a 2′-O-(2-methoxyethyl)(MOE) modification.

In some embodiments, every nucleotides of the oligonucleotide and/or poly-G sequence comprise a 2′-O-(2-methoxyethyl)(MOE) modification.

In some embodiments, the modification of the ribose group comprises a tricyclo-DNA modification.

In some embodiments, every nucleotide of the oligonucleotide and/or poly-G sequence comprise a tricyclo-DNA modification.

In some embodiments, the modification of the ribose group comprises a 2′-deoxy modification.

In some embodiments, each modification of the phosphate group comprises a phosphorothioate, a phosphonoacetate (PACE), a thiophosphonoacetate (thioPACE), an amide, a triazole, a phosphonate, a phosphotriester, or a combination thereof.

In some embodiments, the modification of the phosphate group is phosphorothioate.

In some embodiments, every nucleotide of the oligonucleotide and/or the poly-G sequence comprise a phosphorothioate.

In some embodiments, the oligonucleotide and/or poly-G sequence comprise at least one phosphodiester intemucleotide linkage.

In some embodiments, every intemucleotide linkage in the oligonucleotide and/or poly-G sequence is a phosphodiester internucleotide linkage.

In some embodiments, each modification of the nucleobase comprises a 2-thiouridine, a 4-thiouridine, a N6-methyladenosine, a pseudouridine, a 2,6-diaminopurine, inosine, a thymidine, a 5-methylcytosine, a 5-substituted pyrimidine, an isoguanine, an isocytosine, a halogenated aromatic group, or a combination thereof.

In some embodiments, the modification of the nucleobase group comprises a 5-methylcytosine modification.

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

In some embodiments, a functional moiety is linked to the 5′ end or 3′ end of the oligonucleotide.

In some embodiments, the functional moiety comprises an N-acetylgalactosamine (GalNAc) moiety and/or a hydrophobic moiety.

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

In some embodiments, the steroid selected from the group consisting of cholesterol and Lithocholic acid (LCA).

In some embodiments, the fatty acid selected from the group consisting of Eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA) and Docosanoic acid (DCA).

In some embodiments, the oligonucleotide comprises the formula:

A-B-C, wherein:

- A comprises from about 0 to about 8 modified nucleotides;
- B comprises from about 6 to about 18 deoxyribonucleic acid (DNA) nucleotides and/or DNA-like nucleotides; and
- C comprises from about 0 to about 8 modified nucleotides;

and wherein the overall length of the antisense oligonucleotide is about 10 to about 30 nucleotides.

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

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

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

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

In some embodiments, the oligonucleotide comprises a nucleic acid sequence with at least 90% sequence identity of any one of the nucleic acid sequence of Table 1.

In some embodiments, the oligonucleotide comprises a sequence modification pattern of XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsX, wherein

- s represents a phosphorothioate internucleoside linkage;
- X comprises an adenosine, a guanosine, a cytidine, a thymine, or a uracil, wherein X comprises a 2′-O-(2-methoxyethyl) modification; and
- X comprises an adenosine, a guanosine, a cytidine, a thymine, or a uracil, wherein X comprises a 2′-deoxy modification.

In some embodiments, the oligonucleotide comprises a sequence modification pattern of XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsX, wherein

- s represents a phosphorothioate internucleoside linkage; and
- X comprises an adenosine, a guanosine, a cytidine, a thymine, or a uracil, wherein X comprises a 2′-O-(2-methoxyethyl) modification.

In some embodiments, the oligonucleotide comprises a sequence modification pattern of XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsX, wherein

- s represents a phosphorothioate internucleoside linkage; and
- X comprises an adenosine, a guanosine, a cytidine, a thymine, or a uracil, wherein X comprises a 2′-O-(2-methoxyethyl) modification.

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

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

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

In some embodiments, the target is SOD1.

In some embodiments, the oligonucleotide of comprises the sequence (eC) #(eA)(eG) #(eG)(eA) #(dT) #(dA) #(d5C) #(dA) #(dT) #(dT) #(dT) #(d5C) #(dT) #(dA) #(eC)(e A) #(eG)(eC) #(eU)(polyG)(SEQ ID NO: 48), wherein (#) indicates phosphorothioate linkage, (e) indicates a 2′MOE modification, (d) indicates a deoxyribonucleotide, (dN) indicates a deoxyribonucleotide of A, T, or C, (d5C) indicates 5-methyl cytosine, and (polyG) indicates 2-30 G nucleotides.

In some embodiments, the oligonucleotide of comprises the sequence (eC) #(eA)(eG) #(eG)(eA) #(dT) #(dA) #(d5C) #(dA) #(dT) #(dT) #(dT) #(d5C) #(dT) #(dA) #(eC)(e A) #(eG)(eC) #(eU)(dG)_x(SEQ ID NO: 49), wherein (#) indicates phosphorothioate linkage, (e) indicates a 2′MOE modification, (d) indicates a deoxyribonucleotide, (d5C) indicates 5-methyl cytosine, and x indicates an integer between 5-10.

In some embodiments, the oligonucleotide of comprises the sequence (eC) #(eA)(eG) #(eG)(eA) #(dT) #(dA) #(d5C) #(dA) #(dT) #(dT) #(dT) #(d5C) #(dT) #(dA) #(eC)(e A) #(eG)(eC) #(eU)(dGdGdGdN)_x(SEQ ID NO: 50), wherein (#) indicates phosphorothioate linkage, (e) indicates a 2′MOE modification, (d) indicates a deoxyribonucleotide, (d5C) indicates 5-methyl cytosine, (dN) indicates a deoxyribonucleotide of A, T, or C, and x indicates an integer between 2-10.

In some embodiments, the target is PCSK9.

In some embodiments, the oligonucleotide is selected from the group consisting of an antisense oligonucleotide (ASO), a gapmer, a siRNA, a miRNA, a shRNA, a CRISPR guide, a DNA, an antisense mixmer, a miRNA inhibitor, a splice-switching oligonucleotide (SSO), a phosphorodiamidate morpholino oligomer (PMO), and a peptide nucleic acid (PNA).

In some embodiments, the oligonucleotide is a double-stranded RNA (dsRNA).

In some embodiments, the dsRNA comprises an antisense strand complementary to a 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 that is 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.

In some embodiments, the poly-G sequence is linked to the 5′ end and/or 3′ end of the antisense strand and/or sense strand.

In some embodiments, the target is PCSK9.

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)(m U)(mA)(mG) #(mA) #(mA)(SEQ ID NO: 40) and a sense strand of (mC) #(mU) #(mA)(mG)(mA)(mC)(fC)(mU)(fG)(mU)(dT)(mU)(mU)(mG)(mC)(mU)(mU)(m U)(mU)(mG)(mU)GalNac(SEQ ID NO: 39), wherein (#) indicates a phosphorothioate linkage, (mN) indicates a 2′-OMe modification, (fN) indicates a 2′-Fluoro modification, V indicates 5′-Vinyl phosphate, and GalNac indicates a N-acetylgalactosamine (GalNAc) conjugate.

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)(m U)(mA)(mG) #(mA) #(mA)(polyG)(SEQ ID NO: 51), wherein (#) indicates a phosphorothioate linkage, (mN) indicates a 2′-OMe modification, (fN) indicates a 2′-Fluoro modification, V indicates 5′-Vinyl phosphate, and (polyG) indicates 2-30 G nucleotides.

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)(m U)(mA)(mG) #(mA) #(mA)(dG)_x(SEQ ID NO: 52), wherein (#) indicates a phosphorothioate linkage, (mN) indicates a 2′-OMe modification, (fN) indicates a 2′-Fluoro modification, V indicates 5′-Vinyl phosphate, and x indicates an integer between 5-10.

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

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

In some embodiments, the oligonucleotide or the dsRNA inhibit the expression of the EXOC2 gene, the Ku80 gene, or the Task1 gene by at least about 50%.

In some embodiments, the oligonucleotide or the dsRNA inhibit the expression of the EXOC2 gene, the Ku80 gene, or the Task1 gene by at least about 80%.

In another aspect, the disclosure provides a vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes the oligonucleotide or the dsRNA.

In some embodiments, the oligonucleotide or the dsRNA inhibit the expression of a gene by at least 30%

In some embodiments, the oligonucleotide or the dsRNA inhibit the expression of a gene by at least about 50%.

In some embodiments, the oligonucleotide or the dsRNA inhibit the expression of a gene by at least about 80%.

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

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

In another aspect, the disclosure provides a cell comprising the vector or the rAAV.

In another aspect, the disclosure provides a method for inhibiting expression of a gene in a cell, the method comprising:

- (a) introducing into the cell the oligonucleotide, the dsRNA, the vector, or the rAAV; and
- (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the gene, thereby inhibiting expression of the gene in the cell.

In some embodiments, the poly-G sequence directs the degradation from a lysosome.

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

In some embodiments, the oligonucleotide, the dsRNA, the vector, or the rAAV inhibit the expression of the EXOC2 gene, the Ku80 gene, or the Task1 gene by at least about 50%.

In some embodiments, the oligonucleotide, the dsRNA, the vector, or the rAAV inhibit the expression of the EXOC2 gene, the Ku80 gene, or the Task1 gene by at least about 80%.

In some embodiments, the oligonucleotide, the dsRNA, the vector, or the rAAV is administered by intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof.

In another aspect, the disclosure provides a method of treating or managing a disease associated with a gene comprising administering to a patient in need of such treatment a therapeutically effective amount of the oligonucleotide, the dsRNA, the vector, or the rAAV.

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

In some embodiments, the oligonucleotide, the dsRNA, the vector, or the rAAV inhibit the expression of the EXOC2 gene, the Ku80 gene, or the Task1 gene by at least about 50%.

In some embodiments, the oligonucleotide, the dsRNA, the vector, or the rAAV inhibit the expression of the EXOC2 gene, the Ku80 gene, or the Task1 gene by at least about 80%.

In another aspect, the disclosure provides a method for degrading a target polynucleotide in a lysosome 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 the cell, and maintaining the cell for time sufficient to degrade the target polynucleotide in the lysosome of the cell.

In some embodiments, the method for degrading the target polynucleotide in the lysosome of the cell occurs in vivo, ex vivo, or in vitro.

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

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

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

In another aspect, the disclosure provides a method of treating or managing amyotrophic lateral sclerosis (ALS) in a patient, comprising administering to the patient a therapeutically effective amount of an oligonucleotide with complementarity to SOD1 described herein.

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

In some embodiments, the primary hyperlipidemia is heterozygous familial hypercholesterolemia (HeFH).

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

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present disclosure will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 depicts a schematic of the RNase H and lysosome based knockdown approaches to knockdown a target RNA.

FIG. 2 depicts a schematic of an exemplary Lyso-ASO that can target lysosome. The Gapmer ASO contains 5′/3′ terminal 2′MOE modifications with internal DNA nucleotides. Each intemucleotide linkage in the ASO is a phosphorothioate modification. The lyso sequence is unmodified.

FIG. 3 depicts relative EXOC2 mRNA level as measured by qRT-PCT analysis for gapmers with and without poly-G, poly-A, or poly-C ligand. N=7-9. Values are mean±S.E.M. *p<0.05; **p<0.01; ***p<0.001; **** p<0.0001, by two-tailed Student's t-test.

FIG. 4 (SEQ ID NOs: 56 and 59) depicts relative EXOC2 mRNA level as measured by qRT-PCT analysis for gapmers with and without poly-G ligand of different lengths. N=6-12. Values are mean±S.E.M. *p<0.05; **p<0.01; ***p<0.001; **** p<0.0001, by two-tailed Student's t-test.

FIG. 5 depicts relative EXOC2 mRNA level as measured by qRT-PCT analysis for EXOC2 349. N=3, independent differentiations. Values are mean±S.E.M. *p<0.05; **p<0.01; ***p<0.001; **** p<0.0001, by two-tailed Student's t-test.

FIG. 6 depicts relative EXOC2 mRNA level as measured by qRT-PCT analysis for EXOC2 3933. N=6. Values are mean±S.E.M. *p<0.05; **p<0.01; ***p<0.001; **** p <0.0001, by two-tailed Student's t-test.

FIG. 7 depicts a schematic of the BAFILOMYCIN A1 (Baf A1) inhibition of lysosomal V-ATPase and thus of lysosomal function.

FIG. 8 depicts relative EXOC2 mRNA level as measured by qRT-PCT analysis for EXOC2 3933 with inhibited and un-inhibited lysosome. N=6. Values are mean±S.E.M. *p <0.05; **p<0.01; ***p<0.001; **** p<0.0001, by two-tailed Student's t-test.

FIG. 9 depicts a schematic of the RNase H and lysosome based knockdown approaches to knockdown a target RNA using a sterically blocked ASO.

FIG. 10 depicts relative EXOC2 mRNA level as measured by qRT-PCT analysis for EXOC2 3933 that is sterically blocked and not blocked. N=4-6. Values are mean±S.E.M. *p <0.05; **p<0.01; ***p<0.001; **** p<0.0001, by two-tailed Student's t-test.

FIG. 11 depicts relative Ku80 mRNA level as measured by qRT-PCT analysis for Ku80 624 and 2802. N=6. Values are mean±S.E.M. *p<0.05; **p<0.01; ***p<0.001; **** p <0.0001, by two-tailed Student's t-test.

FIG. 12 depicts relative TASK1 mRNA level as measured by qRT-PCT analysis for TASK1 622 and 5968. N=6. Values are mean±S.E.M. *p<0.05; **p<0.01; ***p<0.001; **** p<0.0001, by two-tailed Student's t-test.

FIG. 13 depicts relative SOD1 mRNA level as measured by qRT-PCT analysis in SH-SY5Y cells treated with 25 or 100 nM Nontargeting control (NTC), Tofersen, or Tofersen-Lamp ASOs for 24 hrs (n=3). Values are mean±S.E.M. *p<0.05; **p<0.01; ***p<0.001; **** p<0.0001, by one-tailed t-test. FIG. 14 depicts relative PCSK9 mRNA level as measured by qRT-PCT analysis in A549 cells treated with 50 nM Inclisiran or Inclisiran-Lamp for 48 hrs (n=4). Values are mean±S.E.M. *p<0.05; **p<0.01; ***p<0.001; **** p<0.0001, by one-tailed t-test.

FIG. 15 depicts relative SOD1 mRNA level as measured by qRT-PCT analysis in SH-SY5Y cells treated with Vehicle (Mock), 30 nM Tofersen-Lamp, or Tofersen-Lamp derivative ASOs for 30 hrs (n=3). Values are mean±S.E.M. *p<0.05; **p<0.01; ***p<0.001; **** p<0.0001, by one- or two-tailed t-test.

DETAILED DESCRIPTION

The present disclosure provides oligonucleotides, methods, and compositions for degrading a target polynucleotide (e.g., an RNA) using the lysosomal pathway. The disclosure described herein is largely based on the finding that antisense compounds conjugated with a lysosome ligand (e.g., a poly-G sequence) facilitate the degradation of a target polynucleotide via the lysosomal pathway. Without being bound to any specific theory, the lysosome ligand from the antisense compound interacts with the LAMP-2C protein of the lysosome and triggers the uptake and degradation by the lysosome of the target polynucleotide attached to the antisense compound (e.g., an antisense oligonucleotide).

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

Furthermore, the experiments described herein, unless otherwise indicated, can use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition) by M R Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2^ndedition).

Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and/or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

So that the disclosure can be more readily understood, certain terms are first defined.

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

The term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refers 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 term “DNA” or “DNA molecule” or “deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleotides. A DNA nucleotide refers to a single deoxyribonucleotide. As used herein, the term “DNA-like” refers to a conformation of, e.g. a modified nucleoside or nucleotide which is similar to the conformation of a corresponding unmodified DNA unit. For example, a DNA-like nucleotide can refer to a conformation of a modified deoxyribonucleotide similar to a corresponding unmodified deoxyribonucleotide. Examples of DNA-like nucleotides include, without limitation, e.g., 2′-deoxyribonucleotides, 2′-deoxy-2′-substituted arabinonucleotides (e.g., 2′-deoxy-2′-fluoroarabinonucleotides, also known in the art as 2′F-ANA or FANA), and corresponding phosphorothioate analogs. As used herein, the term “RNA-like” refers to a conformation of, e.g. a modified nucleoside or nucleotide which is similar to the conformation of a corresponding unmodified RNA unit. RNA-like conformations can adopt an A-form helix while DNA-like conformations adopt a B-form helix. Examples of RNA-like nucleotides include, without limitation, e.g., 2′-substituted-RNA nucleotides (e.g., 2′-fluoro-RNA nucleotides also known in the art as 2′F-RNA), locked nucleic acid (LNA) nucleotides (also known in the art as bridged nucleic acids or bicyclic nucleotides), 2′-fluoro-4′-thioarabinonucleotide (also known in the art as 4'S-FANA nucleotides), 2′-O-alkyl-RNA, and corresponding phosphorothioate analogs.

DNA and RNA can be synthesized naturally (e.g., by DNA replication or transcription of DNA, 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 single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA.

As used herein, the term “small interfering RNA” (“siRNA”)(also referred to in the art as “short interfering RNAs”) refers to an RNA (or RNA analog) comprising between about 10-50 nucleotides (or nucleotide analogs) which is capable of directing or mediating RNA interference. In one embodiment, a siRNA comprises between about 15-30 nucleotides or nucleotide analogs, or between about 16-25 nucleotides (or nucleotide analogs), or between about 18-23 nucleotides (or nucleotide analogs), or between about 19-22 nucleotides (or nucleotide analogs)(e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs). The term “short” siRNA refers to a siRNA comprising about 21 nucleotides (or nucleotide analogs), for example, 19, 20, 21 or 22 nucleotides. The term “long” siRNA refers to a siRNA comprising about 24-25 nucleotides, for example, 23, 24, 25 or 26 nucleotides. Short siRNAs can, in some instances, include fewer than 19 nucleotides, e.g., 16, 17 or 18 nucleotides, provided that the shorter siRNA retains the ability to mediate RNAi. Likewise, long siRNAs can, in some instances, include more than 26 nucleotides, provided that the longer siRNA retains the ability to mediate RNAi absent further processing, e.g., enzymatic processing, to a short siRNA.

The term “nucleotide analog” or “altered nucleotide” or “modified nucleotide” refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary modified nucleotides are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the modified nucleotide to perform its intended function. Examples of positions of the nucleotide which can be derivatized include the 5 position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine, 5-propenyl uridine, etc.; the 6 position, e.g., 6-(2-amino)propyl uridine; the 8-position for adenosine and/or guanosines, e.g., 8-bromo guanosine, 8-chloro guanosine, 8-fluoroguanosine, etc. Modified nucleotides also include deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-modified (e.g., alkylated, e.g., N6-methyl adenosine, or as otherwise known in the art) nucleotides; and other heterocyclically modified nucleotides such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

Modified nucleotides can also comprise modifications to the sugar portion of the nucleotides. 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, wherein R is substituted or unsubstituted C₁-C₆alkyl, alkenyl, alkynyl, aryl, etc. For another example, the ribose sugar can be replaced with a bicyclic or tricylic moiety, such as in Locked Nucleic Acid, constrained ethyl, tricyclo-DNA (tcDNA), or other bridged or bicyclic modifications. Other possible modifications include those described in U.S. Pat. Nos. 5,858,988, and 6,291,438.

The phosphate group of the nucleotide can also be modified, e.g., by substituting one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioates), or by making other substitutions which allow the nucleotide to perform its intended function such as described in Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of the above-referenced modifications (e.g., phosphate group modifications) decrease the rate of hydrolysis of polynucleotides comprising said analogs in vivo or in vitro.

As used herein, the terms “unmodified nucleotide” or “non-modified nucleotide” refers to a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In some embodiments, a non-modified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleoside) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

The term “oligonucleotide” refers to a short polymer of nucleotides and/or modified nucleotides. As discussed above, the oligonucleotides can be linked with non-phosphodiester linkages, which result in a lower rate of hydrolysis as compared to an oligonucleotide linked with phosphodiester linkages. For example, the nucleotides of the oligonucleotide can comprise triazole, amide, carbamate, methylenediol, ethylene diol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoroamidate, phosphonate, and/or phosphorothioate linkages. Alterations or modifications of the oligonucleotide can further include addition of non-nucleotide material to the end(s) of the oligonucleotide or internally (at one or more nucleotides of the oligonucleotide). The oligonucleotide can comprise a lysosome ligand.

As used herein, the term “antisense oligonucleotide” refers to an oligonucleotide molecule, which is capable of binding to RNA inside cells by Watson-Crick base pairing. Depending on the sequence and chemistry of the antisense oligonucleotide, this interaction can lead to silencing of a target gene (i.e. reducing the level of expression of mature mRNA and/or protein from that gene) or activation of a target gene (i.e. increasing the level of expression of mature mRNA and/or protein from that gene). The antisense oligonucleotides of the present disclosure are focused on activating gene expression, which can be done utilizing different mechanisms. Some antisense oligonucleotides are designed to recruit RNase H to cleave their target RNAs. RNase H is a family of non-sequence-specific endonuclease enzymes that catalyze the cleavage of RNA in an RNA/DNA substrate via a hydrolytic mechanism. In certain embodiments, the antisense oligonucleotides of the disclosure trigger RNase H-mediated cleavage of a pre-mRNA target (e.g., EXOC2, Ku80, and Task1 pre-mRNA), which can be compatible with activation of overall target gene expression (e.g., EXOC2, Ku80, and Task1 gene expression). Other antisense oligonucleotides, called steric blockers, are designed not to elicit cleavage of their targets but to block interactions with cellular factors. For example, these cellular factors could modulate splicing, block interactions of noncoding RNAs or of RNA-binding proteins, stabilize mRNA to prolong its half-life, or increase the efficiency of translation of an mRNA.

Antisense oligonucleotides designed to recruit RNase H are often designed as “gapmers.” The term “gapmer” means a chimeric antisense oligonucleotide in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region can be referred to as a “gap segment” and the external regions can be referred to as “wing segments.” “Chimeric antisense oligonucleotide” means an antisense oligonucleotide that has at least two chemically distinct regions.

As used herein, the term “lysosome ligand” is a ligand that interacts with a lysosome. Without being bound to any specific theory, the lysosome ligand interacts with the LAMP-2C protein of the lysosome and triggers the uptake and degradation by the lysosome of a target RNA attached to an oligonucleotide comprising the lysosome ligand. In some embodiments, the lysosome ligand is a poly-G ligand or sequence.

As used herein, a “poly-G ligand” or “poly-G sequence” is a polynucleotide sequence containing two or more G nucleotides and lacks complementarity to the target polynucleotide of the oligonucleotide to which the poly-G sequence is attached.

In some embodiments, the poly-G sequence comprises 2-30 G nucleotides. In some embodiments, the poly-G sequence is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 G nucleotides (SEQ ID NO: 57). 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.

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

In some embodiments, the poly-G sequence comprises G nucleotides that are consecutive. In some embodiments, the poly-G sequence comprises or consists of 5-15 consecutive G nucleotides. In some embodiments, the poly-G sequence comprises 1 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 1 or more non-G nucleotides (e.g., A, T, U, or C) between the two or more poly-G sequences. For example, but in no way limiting, the oligonucleotide may comprise at the 3′ end (from 5′ to 3′), 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.

In some embodiments, the poly-G sequence comprises G nucleotides that are not consecutive.

As used herein, the term “functional moiety” is a moiety that is linked to an oligonucleotide. a functional moiety is linked to the 5′ end or 3′ end of the oligonucleotide. The functional moiety can comprise an N-acetylgalactosamine (GalNAc) moiety and/or a hydrophobic moiety. The hydrophobic moiety can be fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and a mixture thereof. The steroid can be cholesterol and Lithocholic acid (LCA). The fatty acid can be Eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA), and Docosanoic acid (DCA).

As used herein, the term “target polynucleotide” is a polynucleotide which has sufficient complementarity to an oligonucleotide of the disclosure to mediate lysosomal targeting via the poly-G sequence. Exemplary target polynucleotides include mRNA and viral RNA.

As used herein, the term “target gene” is a gene whose expression is to be substantially silenced, restored, or increased. In certain embodiments, the target gene expression is silenced, restored, or increased to wild type levels by an oligonucleotide that silenced expression through direct base pairing interactions with the target sequence and via the lysosomal pathway (e.g., an EXOC2, Ku80, and Task1 mRNA target sequence). In certain embodiments, the target gene expression is silenced, restored, or increased to wild type levels through RNA silencing, e.g., by cleaving a transcript corresponding to a target gene or translational repression of the target gene. Without wishing to be bound by theory, cleavage of a target transcript can increase the levels of productive transcription of the target gene. For example, but in no way limiting, an allele of the target gene can be expressed to form pre-mRNA which can be defective (e.g., contain a nucleotide repeat region that contributes to disease). The target gene expression can be restored by cleaving and degrading the defective pre-mRNA derived from the defective allele, thereby freeing transcriptional machinery to trigger transcription of the non-defective target gene allele. The term “non-target gene” is a gene whose expression is not to be substantially increased, restored, or silenced. For example, a target gene of the present disclosure is EXOC2, Ku80, and Task1, and a non-target gene of the present disclosure is a gene that is not EXOC2, Ku80, and Task1. In one embodiment, the polynucleotide sequences of the target and non-target gene (e.g., mRNA encoded by the target and non-target genes) can differ by one or more nucleotides. In another embodiment, the target and non-target genes can differ by one or more polymorphisms (e.g., Single Nucleotide Polymorphisms or SNPs). In another embodiment, the target and non-target genes can share less than 100% sequence identity. In another embodiment, the target and non-target genes can share less than 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 86%, 85%, 80%, 75%, or 70% sequence identity. In another embodiment, the non-target gene can be a homologue (e.g., an orthologue or paralogue) of the target gene.

The term “antisense activity” means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In some embodiments, antisense activity is an increase in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid. “Antisense compound” means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. As used herein, “antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.

The term “antisense inhibition” means reduction of target nucleic acid levels in the presence of an antisense oligonucleotide having a sequence that is sufficiently complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound. A target nucleic acid can be any nucleic acid. For example, a target nucleic acid of the present disclosure can be a EXOC2, Ku80, Task1, SOD1, or PCSK9 transcript. In certain embodiments, the target nucleic acid is EXOC2, Ku80, Task1, SOD1, or PCSK9 pre-mRNA.

The term “target-recognition sequence” refers to the portion of an antisense compound that recognizes a target nucleic acid. The target-recognition sequence has a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.

The term “conserved region” refers to a portion, or portions, of a nucleic acid sequence that is conserved, i.e. a portion, or portions of the nucleic acid sequence having a similar or identical sequence across species. A conserved region can be computationally identified, e.g., using any sequence alignment software available in the art.

As used herein, the term “sufficiently complementary” means that the antisense compound has a sequence (e.g., an antisense oligonucleotide having a target-recognition sequence), which is sufficient to bind the desired target transcript (e.g., a EXOC2, Ku80, or Task1transcript), and to increase, restore, or silenced expression of the EXOC2, Ku80, or Task1 gene. For example, a target-recognition sequence with at least 90% complementarity to a target nucleic acid sequence (e.g., a portion of a EXOC2, Ku80, or Task1 transcript) can be sufficiently complementary to increase, restore, or silenced expression of the EXOC2, Ku80, or Task1 gene. The term “perfectly complementary” refers to, e.g., a target-recognition sequence with 100% complementarity to a target nucleic acid sequence. Complementary nucleic acid molecules hybridize to each other. The term “hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include an antisense compound and a target nucleic acid.

As used herein, a “region of complementarity” refers to a portion of the antisense oligonucleotide that is complementary to the target transcript (e.g. the EXOC2, Ku80, or Task1 transcript). For example, but in no way limiting, an 18-nucleotide long antisense oligonucleotide can comprise a contiguous 12-nucleotide portion that is complementary to the target transcript. In certain embodiments, the antisense oligonucleotide is complementary to the target transcript over the full length of the antisense oligonucleotide.

As used herein, “administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an antisense compound provided herein) into a patient. The antisense oligonucleotides described herein can be administered to the central nervous system of a patient. The central nervous system includes the brain and spinal cord. Administration methods to the central nervous system include, but not limited to, intrathecal, intraventricular or intrastriatal infusion or delivery and/or any other method of physical delivery described herein or known in the art. Intraventricular infusion can comprise administration using an Ommaya reservoir. In some embodiments, the antisense oligonucleotides described herein can be administered to the patient systemically (such as intravenously, subcutaneously, or intramuscularly). These compounds can be designed to cross into the central nervous system, or to be active in other tissues, such as muscle (including skeletal or heart muscle) or pancreas.

When a disease, or a symptom thereof, is being managed or treated, the administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease, or symptom thereof, is being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof and can be continued chronically to defer or reduce the appearance or magnitude of disease-associated symptoms, e.g., damage to the involved tissues and airways.

As used herein, the term “composition” is intended to encompass a product containing the specified ingredients (e.g., an antisense compound provided herein) in, optionally, the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in, optionally, the specified amounts.

“Effective amount” means the amount of active pharmaceutical agent (e.g., an antisense compound of the present disclosure) sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount can vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.

As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, a subject can be a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkey and human). In certain embodiments, the term “subject” refers to a vertebrate, such as a mammal. Mammals include, without limitation, humans, non-human primates, wild animals, feral animals, farm animals, sports animals, and pets. In one embodiment, the subject is a mammal, such as a human, having a EXOC2-related disorder, a Ku80-related disorder, and/or a Task1-related disorder. In another embodiment, the subject is a mammal, such as a human, that is at risk for developing a EXOC2-related disorder, aKu80-related disorder, and/or a Task1-related disorder.

As used herein, the term “therapy” refers to any protocol, method and/or agent that can be used in the prevention, management, treatment and/or amelioration of a disease or a symptom related thereto, such as a EXOC2-related disorder, aKu80-related disorder, and/or a Task1-related disorder. In some embodiments, the term “therapy” refers to any protocol, method and/or agent that can be used in the modulation of an immune response to an infection in a subject or a symptom related thereto. In some embodiments, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and/or other therapies useful in the prevention, management, treatment and/or amelioration of a disease or a symptom related thereto, such as a EXOC2-related disorder, a Ku80-related disorder, and/or a Task1-related disorder known to one of skill in the art such as medical personnel. In other embodiments, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and/or other therapies useful in the modulation of an immune response to an infection in a subject or a symptom related thereto known to one of skill in the art.

As used herein, the terms “treat,” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and/or duration of a disease or a symptom related thereto, such as a EXOC2-related disorder, a Ku80-related disorder, and/or a Task1-related disorder, resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents, such as an antisense oligonucleotide provided herein). The term “treating,” as used herein, can also refer to altering the disease course of the subject being treated. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptom(s), diminishment of direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

As used herein, the term “EXOC2” refers to a gene that encodes the protein exocyst complex component 2. The protein EXOC2 is a component of the exocyst complex, a multi-protein complex essential for the polarized targeting of exocytic vesicles to specific docking sites on the plasma membrane. The human EXOC2 gene corresponds to NG_047166.1 of the NCBI RefSeq database.

As used herein, the term “Ku80” refers to a protein encoded by the XRCC5 gene. Ku80 is a component of the Ku heterodimer with Ku70, which binds to DNA double-strand break ends and is required for the non-homologous end joining (NHEJ) pathway of DNA repair. The human XRCC5 gene corresponds to NG_029780.1 of the NCBI RefSeq database. The term Ku80 is used herein to also refer to the gene and mRNA encoding Ku80, Therefore, oligonucleotides that target Ku80, target the Ku80 mRNA (including pre-mRNA).

As used herein, the term “Task1,” or “KCNK3” refers to a gene that encodes the potassium channel subfamily K member 3 protein. Task1 is one of the members of the superfamily of potassium channel proteins containing two pore-forming P domains. The human KCNK3 gene corresponds to NG_033884.1 of the NCBI RefSeq database. Inhibitors of SOD1, such as inclisiran, are used for the treatment of hyperlipidemia or reducing low density lipoprotein cholesterol (LDL-C) in a subject.

As used herein, the term “SOD1” refers to a gene that encodes the superoxide dismutase 1 protein. SOD1 is a mammalian enzyme that catalyzes the removal of superoxide radicals. The human SOD1 gene corresponds NG_008689.1 of the NCBI RefSeq database. Inhibitors of SOD1, such as tofersen, are used for the treatment of amyotrophic lateral sclerosis (ALS) in a subject.

As used herein, the term “PCSK9” refers to a gene that encodes the proprotein convertase subtilisin/kexin type 9 protein. PCSK9 binds to and degrades the receptor for low-density lipoprotein particles (LDL). The human PCSK9 gene corresponds to NG_009061.1 of the NCBI RefSeq database. Inhibitors of PCSK9, such as inclisiran, are used for the treatment of hyperlipidemia or reducing low density lipoprotein cholesterol (LDL-C) in a subject.

Antisense Compounds

The present disclosure provides an antisense compound that linked to a poly-G sequence as disclosed here, for directing a target polynucleotide to the lysosome of a cell for target degradation.

The present disclosure also provides an antisense compound that is capable of decreasing EXOC2, Ku80, and Task1 gene expression 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.

In certain embodiments, the antisense compounds that are capable of decreasing a target polynucleotide expression (e.g., EXOC2, Ku80, and Task1 gene expression), have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced the activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.

In some embodiments, an antisense compound of the present disclosure is an antisense oligonucleotide. Chimeric antisense oligonucleotides typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased activity. A second region of a chimeric antisense compound can optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex. In some embodiments, an antisense compound of the present disclosure is a chimeric antisense oligonucleotide having a gapmer motif In a gapmer, an internal region having a plurality of nucleotides that supports RNase H cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region.

In some embodiments, the present disclosure provides an antisense oligonucleotide having a target-recognition sequence that is sufficiently complementary to an EXOC2, Ku80, and/or Task1 transcript or portion thereof, to direct cleavage of the EXOC2, Ku80, and Task1 transcript by RNase H. 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 comprises a gapmer motif.

In the case of an antisense compound having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer can in some embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides can include 2′-MOE, and 2′-O-CH₃(i.e., OMe), among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides can include those having a 4′-(CH₂)_n-O-2′ bridge, where n=1 or n=2). In some embodiments, the wing segments of the gapmer contain one or more tricyclo-DNA (tcDNA) modifications. In some embodiments, each distinct region comprises uniform sugar moieties. In some embodiments, each wing segment comprises a mixture of different nucleotide modifications. For example, in one embodiment, a LNA modification and a 2′-MOE modification could be used in combination for one antisense compound. In one embodiment, a LNA modification and a 2′-O-Methyl modification could be used in combination for one antisense compound. In one embodiment, a LNA modification and a 2′-deoxy modification could be used in combination for one antisense compound. In one embodiment, a LNA modification and a tricyclo-DNA modification could be used in combination for one antisense compound. In one embodiment, a 2′-MOE modification and a tricyclo-DNA modification could be used in combination for one antisense compound.

The gapmer motif can be described using the formula “A-B-C”, where “A” represents the length of the 5′ wing region, “B” represents the length of the gap region, and “C” represents the length of the 3′ wing region. As such, in some embodiments, an antisense oligonucleotide of the present disclosure has the formula:

A-B-C.

As used herein, a gapmer described as “A-B-C” has a configuration such that the gap segment is positioned immediately adjacent each of the 5′ wing segment and the 3′ wing segment. Thus, no intervening nucleotides exist between the 5′ wing segment and gap segment, or the gap segment and the 3′ wing segment.

In some embodiments, the 5′ wing region represented by “A” comprises from about 0 to about 8 modified nucleotides, e.g., from about 1 to about 6 modified nucleotides. For example, the 5′ wing region represented by “A” can be 0, 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length. In some embodiments, the 3′ wing region represented by “C” comprises about 0 to about 8 modified nucleotides, e.g., from about 1 to about 6 modified nucleotides. For example, the 3′ wing region represented by “C” can be 0, 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length. In some embodiments, “A” and “C” are the same, in some embodiments, they are different.

In some embodiments, the gap region represented by “B” comprises from about 5 to about 18 DNA nucleotides and/or DNA-like nucleotides, e.g., from about 5 to about 12 DNA nucleotides and/or DNA-like nucleotides. For example, the gap region represented by “B” can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 DNA nucleotides and/or DNA-like nucleotides in length. Thus, an antisense oligonucleotide of the present disclosure having a target-recognition sequence with the formula “A-B-C” include, but are not limited to the following gapmer formats, for example 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 can also be of different lengths, such as 1-10-6, 3-9-5, 7-9-2, 4-10-5, or other asymmetric combinations of wing lengths flanking a central DNA gap. In certain embodiments, the gapmer of “A-B-C” is at least 12 nucleotides in length. In certain embodiments, “B” is at least 6 nucleotides in length. A person of skill in the art will be able to identify additional asymmetric combinations of wing lengths.

In certain embodiments, antisense compounds targeted a EXOC2, Ku80, or Task1 nucleic acid possess a 5-9-4 gapmer format. In some embodiments, the antisense compound is an antisense oligonucleotide having a target-recognition sequence with the 5-9-4 format that is sufficiently complementary to a EXOC2, Ku80, or Task1 transcript, or a portion thereof, to direct cleavage of the EXOC2, Ku80, or Task1 transcript by RNase H. In some embodiments, the target-recognition sequence has the formula “A-B-C”, 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 “A-B-C”, 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 “A-B-C”, wherein “A” comprises 2 to 6 2′-O-(2-methoxyethyl)(MOE) modified nucleotides, “B” comprises 6 to 12 DNA nucleotides and/or DNA-like nucleotides, and “C” comprises 2 to 6 2′-O-(2-methoxyethyl)(MOE) modified nucleotides. In some embodiments, the target-recognition sequence has the formula “A-B-C”, wherein “A” comprises 5 2′-O-(2-methoxyethyl)(MOE) modified nucleotides, “B” comprises 9 DNA nucleotides and/or DNA-like nucleotides, and “C” comprises 4 2′-O-(2-methoxyethyl)(MOE) modified nucleotides.

In some embodiments, antisense compounds that target a EXOC2, Ku80, or Task1 nucleic acid possess a “wingmer” motif. The wingmer motif can be described using the formula “X-Y” or “Y-X”, where “X” represents the length of the wing region, and “Y” represents the length of the gap region. As such, in some embodiments, an antisense oligonucleotide of the present disclosure has the formula:

X-Y, or

Y-X.

As used herein, a wingmer described as “X-Y” or “Y-X” has a configuration such that the gap segment is positioned immediately adjacent to the wing segment. Thus, no intervening nucleotides exist between the wing segment and the gap segment. Non-limiting examples of wingmer configurations of an antisense compound of the present disclosure include, e.g., 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.

In some embodiments, antisense compounds targeted to a EXOC2, KU80, or TASK1 nucleic acid possess a gap-widened motif. As used herein, “gap-widened” refers to an antisense compound having a gap segment of 12 or more contiguous DNA nucleotides and/or DNA-like nucleotides adjacent to a wing region. In the case of a gap-widened gapmer, the gapmer comprises a gap region having 12 or more contiguous DNA nucleotides and/or DNA-like nucleotides positioned between and immediately adjacent to the 5′ and 3′ wing segments. In the case of a gap-widened wingmer, the wingmer comprises a gap region having 12 or more contiguous DNA nucleotides and/or DNA-like nucleotides positioned immediately adjacent to the wing segment.

A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.

Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides can also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.

The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.

In certain embodiments, antisense compounds targeted to a EXOC2, KU80, or TASK1 nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.

Antisense compounds of the disclosure can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar-modified nucleosides can impart enhanced nuclease stability, increased binding affinity or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise a chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include without limitation, addition of substituent groups (including 5′ and 2′ substituent groups, bridging of ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(R¹)(R²)(R═H, C₁-C₁₂alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2′-F-5′-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a BNA (see PCT International Application WO 2007/134181 Published on Nov. 22, 2007 wherein LNA is substituted with for example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 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) substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, 0-allyl, 0-C1-C10 alkyl, OCF₃, O(CH₂)₂SCH₃, O(CH₂)₂-O-N(R_m)(R_n), and O-CH₂-C(═O)—N(R_m)(R_n), where each R_mand R_nis, independently, H or substituted or unsubstituted C₁-C₁₀alkyl. 2′-modified nucleotides are useful in the present disclosure, for example, 2′-O-methyl RNA, 2′-0-methoxyethyl RNA, 2′-fluoro RNA, and others envisioned by one of ordinary skill in the art.

Examples of bicyclic nucleic acids (BNAs) include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. A BNA comprising a bridge between the 4′ and 2′ ribosyl ring atoms can be referred to as a locked nucleic acid (LNA), and is often referred to as inaccessible RNA. As used herein, the term “locked nucleotide” or “locked nucleic acid (LNA)” comprises nucleotides in which the 2′ deoxy ribose sugar moiety is modified by introduction of a structure containing a heteroatom bridging from the 2′ to the 4′ carbon atoms. The term “non-locked nucleotide” comprises nucleotides that do not contain a bridging structure in the ribose sugar moiety. Thus, the term comprises DNA and RNA nucleotide monomers (phosphorylated adenosine, guanosine, uridine, cytidine, deoxyadenosine, deoxyguanosine, deoxythymidine, deoxycytidine) and derivatives thereof as well as other nucleotides having a 2′-deoxy-erythro-pentofuranosyl sugar moiety or a ribo-pentofuranosyl moiety. In certain embodiments, antisense compounds provided herein include one or more BNA nucleosides wherein the bridge comprises one of the 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. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-CH₂-N(OCH₃)-2′ (see PCT/US2008/064591); 4′-CH₂-O-N(CH₃)-2′ (see published U.S. Patent Application US2004-0171570, published Sep. 2, 2004); 4′-CH₂-N(R)-O-2′ (see U.S. Pat. No. 7,427,672, issued on Sep. 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 a protecting group. Each of the foregoing BNAs include various stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).

In some embodiments, antisense compounds provided herein include one or more 2′, 4′-constrained nucleotides. For example, antisense compounds provided by the present disclosure include those having one or more constrained ethyl (cEt) or constrained methoxyethyl(cMOE) nucleotides. In some embodiments, antisense compounds provided herein are antisense oligonucleotides comprising one or more constrained ethyl (cEt) nucleotides. The terms “constrained ethyl” and “ethyl-constrained” are used interchangeably.

In certain embodiments, nucleosides are modified by replacement of the ribosyl ring with a sugar surrogate. Such modification includes without limitation, replacement of the ribosyl ring with a surrogate ring system (sometimes referred to as DNA analogs) such as a morpholino ring, a cyclohexenyl ring, a cyclohexyl ring or a tetrahydropyranyl ring such as one having one of the formula:

embedded image

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

Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that 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, K. R.; Obika, S., Recent Advances in Medicinal Chemistry of Antisense Oligonucleotides. In Comprehensive Medicinal Chemistry, 3rd edition, Elsevier: 2017). Such ring systems can undergo various additional substitutions to enhance activity.

Methods for the preparations of 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) are maintained for hybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds targeted to a EXOC2, Ku80, or Task1 nucleic acid comprise one or more kinds of modified nucleotides. In one embodiment, antisense compounds targeted to a EXOC2, Ku80, or Task1 nucleic acid comprise 2′-modified nucleotides. In one embodiment, antisense compounds targeted to a EXOC2, Ku80, or Task1 nucleic acid comprise a 2′-O-methyl RNA, a 2′-O-methoxyethyl RNA, or a 2′-fluoro RNA. In one embodiment, antisense compounds targeted to a EXOC2, Ku80, or Task1 nucleic acid comprise tricyclo-DNA (tcDNA). Tricyclo-DNA belongs to a class of constrained DNA analogs that display improved hybridizing capacities to complementary RNA, see, e.g., 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, antisense compounds targeted to a EXOC2, Ku80, or Task1 nucleic acid comprise a locked nucleotide, an ethyl-constrained nucleotide, or an alpha-L-locked nucleic acid. Various alpha-L-locked nucleic acids are known by those of ordinary skill in the art, and are described in, e.g., Sorensen et al., J Am. Chem. Soc. (2002) 124(10):2164-2176.

In certain embodiments, the antisense compounds targeting a EXOC2, Ku80, or Task1 nucleic acid are fully chemically modified, i.e., every nucleotide is chemically modified. In certain embodiments, every nucleotide comprises a 2′-O-(2-methoxyethyl)(MOE) modification. In certain embodiments, every nucleotide comprises a tricyclo-DNA modification. In certain embodiments, the antisense compounds targeting a EXOC2, Ku80, or Task1 nucleic acid comprise a mixture of tricyclo-DNA modifications and 2′-O-(2-methoxyethyl)(MOE) modifications, wherein every nucleotide of the antisense compounds is either tcDNA or MOE.

Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

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

Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, antisense compounds targeted to a EXOC2, Ku80, or Task1 nucleic acid comprise one or more modified nucleotides having modified sugar moieties. In some embodiments, the modified nucleotide is a locked nucleotide. In certain embodiments, the locked nucleotides are arranged in a gapmer motif, e.g. a 3-9-3 gapmer format wherein 9 non-locked nucleotides are flanked by 3 locked nucleotides on each side. In certain embodiments, antisense compounds targeted to a EXOC2, Ku80, or Task1 nucleic acid comprise one or more modified nucleotides. In some embodiments, the modified nucleotide is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine. In some embodiments, the modified nucleotide is a 2′-O-(2-methoxyethyl)(MOE) modified nucleotide. In certain embodiments, the 2′-O-(MOE) modified nucleotides are arranged in a gapmer motif, e.g. a 5-9-4 gapmer format wherein 9 non-2′-O-(MOE) modified nucleotides are flanked by 4 or 5 2′-O-(MOE) modified nucleotides on one or both sides. In certain embodiments, antisense compounds targeted to a EXOC2, Ku80, or Task1 nucleic acid comprise a steric blocking chemical modification format. In some embodiments of the steric blocking chemical modification format, every nucleotide of the antisense compound is a 2′-O-(2-methoxyethyl) (MOE) modified nucleotide. In some embodiments of the steric blocking chemical modification format, every nucleotide of the antisense compound is a tricyclo-DNA modified nucleotide. In some embodiments of the steric blocking chemical modification format, the antisense compound comprises at least one MOE modified nucleotide and at least one tricyclo-DNA modified nucleotide. Many different chemical modification patterns steric blocking antisense oligonucleotides are envisioned. For example, but in no way limiting, the steric blocking antisense oligonucleotide can comprise a mixture of different types of modifications, such as a mixture of 2′-O-(2-methoxyethyl) modifications, LNA modifications, tricyclo-DNA modifications, and DNA modifications where the DNA stretches are four nucleotides or less.

In some embodiments, an antisense compound of the present disclosure directs cleavage of an EXOC2, Ku80, or Task1 transcript by RNase H. In such embodiments, the antisense compound can 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, an antisense oligonucleotide of the present disclosure is an RNase H-dependent antisense oligonucleotide, and can be a single-stranded, chemically modified oligonucleotide that binds to a complementary sequence in the target transcript (e.g., a EXOC2, Ku80, or Task1 transcript). An RNase H-dependent antisense oligonucleotide of the present disclosure reduces expression of a target gene by RNase H-mediated cleavage of the target transcript, and by inhibition of translation by steric blockade of ribosomes. In some embodiments, an antisense compound of the present disclosure is capable of mediating cleavage of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more of EXOC2, Ku80, or Task1 transcripts by RNase-H. In one embodiment, the antisense compound is capable of mediating cleavage of at least 80% of EXOC2, Ku80, or Task1 transcripts by RNase-H. In one embodiment, the antisense compound is capable of mediating cleavage of at least 90% of EXOC2, Ku80, or Task1 transcripts by RNase-H.

In certain embodiments, an antisense compound that targets a EXOC2, Ku80, or Task1 transcript is from about 6 to about 24 subunits in length. In other embodiments, the antisense compound that targets an EXOC2, Ku80, or Task1 transcript is from about 8 to about 80 subunits in length. For example, the antisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In some embodiments, the antisense compounds are less than 40 linked subunits in length. In some embodiments, the antisense compounds are from about 10 to about 30 linked subunits in length. In some embodiments, the antisense compounds are from about 12 to about 25 linked subunits in length. In some embodiments, the antisense compounds are from about 15 to about 20 linked subunits in length. In some embodiments, the antisense compound is an antisense oligonucleotide that targets a EXOC2, Ku80, or Task1 transcript, and the linked subunits are linked nucleotides.

In certain embodiments antisense compounds targeted to an EXOC2, Ku80, or Task1 transcript can be shortened or truncated. For example, a single subunit can be deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated antisense compound targeted to a EXOC2, Ku80, or Task1 transcript can have two subunits deleted from the 5′ end, or alternatively can have two subunits deleted from the 3′ end, of the antisense compound. Alternatively, the deleted nucleosides can be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5′ end and one nucleoside deleted from the 3′ end.

When a single additional subunit is present in a lengthened antisense compound, the additional subunit can be located at the 5′ or 3′ end of the antisense compound. When two or more additional subunits are present, the added subunits can be adjacent to each other, for example, in an antisense compound having two subunits added to the 5′ end (5′ addition), or alternatively to the 3′ end (3′ addition), of the antisense compound. Alternatively, the added subunits can be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5′ end and one subunit added to the 3′ end.

It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.

In certain embodiments, the antisense oligonucleotide comprises the formula:

A-B-C, wherein:

- A comprises from about 0 to about 18 modified nucleotides;
- B comprises from about 0 to about 4 deoxyribonucleic acid (DNA) nucleotides and/or DNA-like nucleotides; and
- C comprises from about 0 to about 18 modified nucleotides;
- and the overall length of the antisense oligonucleotide is about 10 to about 30 nucleotides.
  
  Antisense oligonucleotides that contain 4 or fewer DNA and/or DNA-like nucleotides in “B” should not recruit RNase H and direct cleavage of a target. In these instances, the antisense oligonucleotide is not a gapmer format, but can rather act as a steric blocker.

Branched Antisense Compounds

The present disclosure also provides branched antisense compounds comprising two or more target-recognition sequences that targets a portion of a EXOC2, Ku80, or Task1 nucleic acid. A branched antisense compound of the present disclosure can be, e.g., a branched antisense oligonucleotide compound.

As used herein, the term “branched antisense compound” or “branched antisense oligonucleotide” refers to two or more antisense compounds or antisense oligonucleotides that are connected together.

In one embodiment, a branched oligonucleotide compound comprises two or more target-recognition sequences, wherein the target-recognition sequences are connected to one another by one or more moieties selected from a linker, a spacer, and a branching point. 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 sequences comprises a 5′ end and a 3′ end, and each target-recognition sequence is independently connected to a linker, a spacer, or a branching point at the 5′ end or the 3′ end. In some embodiments, each target-recognition sequence is connected to a linker, a spacer, or a branching point at the 5′ end. In some embodiments, each target-recognition sequence is connected to a linker, a spacer, or a branching point at the 3′ end. In another embodiment, each target-recognition sequence is connected to a linker, a spacer, or a branching point. In some embodiments, each of the target-recognition sequences are antisense compounds and/or oligonucleotides that target a portion of a EXOC2, Ku80, or Task1 nucleic acid.

In some embodiments, a branched oligonucleotide compound of the present disclosure has the formula

L-(N)_n

wherein N represents a target-recognition sequence of the present disclosure; n represents an integer, e.g., 2, 3, 4, 5, 6, 7, or 8; and L represents a linker selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and any combination thereof.

In some embodiments, a branched oligonucleotide compound of the present disclosure has the formula

L-(N)_n

wherein the compound optionally further comprises one or more branching points B, and wherein the compound optionally further comprises one or more spacers S. In such embodiments, each of the one or more branching points B independently represents a polyvalent organic species or derivative thereof, and each of the one or more spacers S is independently selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and any combination thereof. In some embodiments, the spacer S is biocleavable. For example, the spacer S could include moieties susceptible to cleavage by nucleases, by proteases, by changes in pH, or by reduction or oxidation. In such embodiments, spacers could include peptides, phosphodiester-linked nucleotides, disulfide bonds, pH-sensitive linkages or other biocleavable moieties. For example, a branched oligonucleotide compound of the present disclosure having the formula L-(N)n has a structure, not to be limited in any fashion, e.g.,

embedded image

Target-Recognition Sequences

The present disclosure provides an antisense oligonucleotide comprising a target-recognition sequence that targets a portion of a EXOC2, Ku80, or Task1 nucleic acid. In certain embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of a portion of a EXOC2, Ku80, or Task1 nucleic acid. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of a portion of a EXOC2, Ku80, or Task1 nucleic acid.

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

Targeting includes determination of at least one target segment to which an antisense oligonucleotide hybridizes, such that a desired effect occurs. In certain embodiments, the desired effect is an increase in transcript target nucleic acid levels, i.e., an increase in EXOC2, Ku80, or Task1 transcript levels. In certain embodiments, the desired effect is an increase of levels of protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid, e.g., an increase in the level of EXOC2, Ku80, or Task1 protein.

A target region can contain one or more target segments. Multiple target segments within a target region can be overlapping. Alternatively, they can be non-overlapping. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain embodiments, target segments within a target region are separated by a number of nucleotides that is about 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or is a range defined by any two of the preceding values. In certain embodiments, target segments within a target region are separated by no more than about 5 nucleotides on the target nucleic acid. In certain embodiments, target segments are contiguous.

Suitable target segments can be found within a 5′ UTR, a coding region, a 3′ UTR, an intron, an exon, and/or an exon/intron junction. Target segments containing a start codon or a stop codon are also suitable target segments. A suitable target segment can specifically exclude a certain structurally defined region such as the start codon or stop codon. In certain embodiments, the target segments are found in intron 1 of the EXOC2, Ku80, and Task1 gene. In certain embodiments, the target segments are found upstream (5′) of a nucleotide repeat region in intron 1 of the EXOC2, Ku80, and Task1 gene. In certain embodiments, the target segments are found downstream (3′) of a nucleotide repeat region in intron 1 of the EXOC2, Ku80, and Task1 gene. In certain embodiments, the target segments are found upstream or downstream of a nucleotide repeat region in intron 1 of the EXOC2, Ku80, and Task1 gene and are unique in a human genome (i.e., the target segment nucleic acid sequence is found only once in the human genome).

The determination of suitable target segments can include a comparison of the sequence of a target nucleic acid (e.g., EXOC2, Ku80, and Task1) to other sequences throughout the genome. For example, the BLAST algorithm can be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense oligonucleotide sequences that can hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences). The determination of suitable target segments can include comparison of the sequences of a target nucleic acid (e.g., a EXOC2, Ku80, and Task1 transcript) across several species. For example, various sequence alignment software can be used to identify regions of similar or identical sequence across species. In certain embodiments, the EXOC2, Ku80, and Task1 transcript target segment nucleic acid sequences are unique in the human genome (i.e., the target segment nucleic acid sequence is found only once in the human genome).

An antisense oligonucleotide and a target nucleic acid (e.g., a EXOC2, Ku80, and Task1transcript or portion thereof) are complementary to each other when a sufficient number of nucleobases of the antisense oligonucleotide can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., increased expression of a target nucleic acid, such as a EXOC2, Ku80, and Task1 transcript or portion thereof).

Non-complementary nucleobases between an antisense oligonucleotide and a EXOC2, Ku80, and Task1 nucleic acid can be tolerated provided that the antisense oligonucleotide remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense oligonucleotide can hybridize over one or more segments of a EXOC2, Ku80, and Task1 nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).

In certain embodiments, the antisense oligonucleotides provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a EXOC2, Ku80, and Task1 nucleic acid, a target region, target segment, or specified portion thereof.

For example, an antisense oligonucleotide in which 18 of 20 nucleobases of the antisense oligonucleotide are complementary to a target region (e.g., an equal length portion of a EXOC2, Ku80, and Task1 transcript), and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases can be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense oligonucleotide which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present disclosure. Percent complementarity of an antisense oligonucleotide with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).

In certain embodiments, the antisense oligonucleotides provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, antisense oligonucleotide can be fully complementary to a EXOC2, Ku80, or Task1 nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” or “perfectly complementary” means each nucleobase of an antisense oligonucleotide is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense oligonucleotide is perfectly complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense oligonucleotide. Perfectly complementary can also be used in reference to a specified portion of the first and/or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense oligonucleotide can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense oligonucleotide. At the same time, the entire 30 nucleobase antisense oligonucleotide may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense oligonucleotide are also complementary to the target sequence.

In one aspect, the disclosure provides an antisense oligonucleotide comprising a region of complementarity to an intron of a EXOC2, Ku80, and Task1 transcript, wherein the antisense oligonucleotide does not comprise a region of complementarity to another site in a human genome (i.e., the target sequence of the antisense oligonucleotide is unique in a human genome).

In another aspect, the disclosure provides an antisense oligonucleotide comprising a region of complementarity to intron 1 of a EXOC2, Ku80, and Task1 transcript, wherein the antisense oligonucleotide does not comprise a region of complementarity to another site in a human genome (i.e., the target sequence of the antisense oligonucleotide is unique in a human genome).