Oligonucleotide-mediated knockdown of GLIS family zinc finger 2 (GLIS2)

Abstract

Provided herein are oligonucleotides for inhibiting the expression of GLIS2 and methods of using the oligonucleotides for reducing GLIS2 expression in a subject.

Description

Attorney Docket No. 63578-717601OLIGONUCLEOTIDE-MEDIATED KNOCKDOWN OF GLIS FAMILY ZINC FINGER 2 (GLIS2)CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 697,320 filed on September 20, 2024, the entirety of which is incorporated herein by reference.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 63578-717_601_SL.xml, created September 18, 2025, which is 15,629,437 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.BACKGROUND

[0003] GLIS2 encodes one of three Gli-similar (Glisl-3) Kriippel-like zinc finger transcription factor proteins. GLIS2 is most abundantly expressed in the kidney along the entire nephron and is the causative gene for nephronophthisis type 7 (NPHP7).

[0004] Polycystic kidney disease (PKD) is a genetic disorder that causes fluid-filled cysts to grow in the kidneys. Autosomal dominant polycystic kidney disease (ADPKD) is a highly penetrant inherited PKD which causes cysts and deformation of the kidneys, typically over the span of decades, and eventually leads to kidney failure requiring dialysis or transplantation in the majority of patients after the fifth decade of life.

[0005] Although ADPKD is sometimes considered an orphan disease, the number of ADPKD patients is in fact significant. There are estimated to be over 600,000 affected individuals with ADPKD in the US alone and over 12 million worldwide. Furthermore, since ADPKD is not subject to founder mutations but rather de novo mutations that occur all the time, the population of ADPKD patients is expected to further grow as the world population expands.SUMMARY

[0006] In one aspect, provides herein is an oligonucleotide for inhibiting expression of GLIS family zinc finger 2 (GLIS2), wherein the oligonucleotide comprises an antisense strand comprising at least 14 contiguous nucleotides substantially complementary to a sequence of nucleotides encoding GLIS2, with no more than 4 mismatched nucleotides.

[0007] In some embodiments, the antisense strand is completely complementary to the sequence of nucleotides encoding GLIS2. In some embodiments, the sequence of nucleotides encodingAttorney Docket No. 63578-717601GLIS2 comprises a nucleotide sequence having at least 90% nucleotide sequence identity to any one of SEQ ID NOs: 2053-2054. In some embodiments, the antisense strand is substantially complementary to a sequence of nucleotides encoding GLIS2. In some embodiments, the antisense strand is substantially complementary to a sequence of nucleotides corresponding to an untranslated region of the GLIS2 transcript. In some embodiments, the antisense strand is completely complementary to a sequence of nucleotides corresponding to an untranslated region of the GLIS2 transcript.In some embodiments, the oligonucleotide is an RNAi agent. In some embodiments, the oligonucleotide is a double stranded small interfering RNA, a short hairpin RNA, or a Dicersubstrate siRNA (DsiRNA). In some embodiments, the oligonucleotide is a double-stranded small interfering RNA (siRNA) further comprising a sense strand, wherein the sense strand and antisense strand comprise a double stranded region.

[0008] In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides differing by no more than 4 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 514-1026, or any one of SEQ ID NOs: 514-1026 or 2119- 2182, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of any one of SEQ ID NOs: 514-1026, or any one of SEQ ID NOs: 514- 1026 or 2119-2182. In some embodiments, the antisense strand differs by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 514-1026, or any one of SEQ ID NOs: 514-1026 or 2119-2182. In some embodiments, the antisense strand differs by no more than 2 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 514-1026, or any one of SEQ ID NOs: 514-1026 or 2119-2182. In some embodiments, the antisense strand differs by no more than 1 nucleotide from the nucleotide sequence of any one of SEQ ID NOs: 514-1026, or any one of SEQ ID NOs: 514-1026 or 2119-2182. In some embodiments, the antisense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 514-1026, or any one of SEQ ID NOs: 514-1026 or 2119-2182.In some embodiments, the sense strand comprises a nucleotide sequence differing by no more than 4 nucleotides from the nucleotide sequence of any one of SEQ ID Nos: 1-513, or any one of SEQ ID NOs: 1-513 or 2055-2118, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of any one of SEQ ID NOs: 1-513, or any one of SEQ ID NOs: 1-513 or 2055-2118.

[0009] In some embodiments, the sense strand differs by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 1-513, or any one of SEQ ID NOs: 1-513 or 2055-2118. In some embodiments, the sense strand differs by no more than 2 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 1-513, or any one of SEQ ID NOs: 1-513 orAttorney Docket No. 63578-7176012055-2118. In some embodiments, the sense strand differs by no more than 1 nucleotide from the nucleotide sequence of any one of SEQ ID NOs: 1-513, or any one of SEQ ID NOs: 1-513 or 2055-2118. In some embodiments, the sense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 1-513, or any one of SEQ ID NOs: 1-513 or 2055-2118.

[0010] In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides differing by no more than 4 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 514-1026, or any one of SEQ ID NOs: 514-1026 or 2119- 2182, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of any one of SEQ ID NOs: 514-1026, or any one of SEQ ID NOs: 514- 1026 or 2119-2182, and the sense strand comprises a nucleotide sequence at least 15 contiguous nucleotides differing by no more than 4 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 1-513, or any one of SEQ ID NOs: 1-513 or 2055-2118, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of any one of SEQ ID NOs: 1-513, or any one of SEQ ID NOs: 1-513 or 2055-2118.

[0011] In some embodiments, each strand is no more than 30 nucleotides in length. In some embodiments, at least one strand comprises a 3’ overhang of at least 1 nucleotide. In some embodiments, at least one strand comprises a 3’ overhang of at least 2 nucleotides. In some embodiments, at least one strand comprises a 5’ overhang of at least 1 nucleotide. In some embodiments, at least one strand comprises a 5’ overhang of at least 2 nucleotides. In some embodiments, the double stranded region is 15-30 nucleotide pairs in length. In some embodiments, the double stranded region is 15-23 nucleotide pairs in length. In some embodiments, the double stranded region is 17-25 nucleotide pairs in length.

[0012] In some embodiments, the double stranded region is 19-23 nucleotide pairs in length. In some embodiments, the double stranded region is 19-21 nucleotide pairs in length. In some embodiments, each strand is 19-30 nucleotides in length. In some embodiments, each strand is 19-23 nucleotides in length. In some embodiments, each strand is 19-21 nucleotides in length. In some embodiments, the antisense is 21 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments, the oligonucleotide is a single-stranded antisense oligonucleotide (ASO). In some embodiments, the ASO comprises a nucleic acid sequence comprising at least 14 contiguous nucleotides that differ by no more than 1, 2, 3, or 4 nucleotides from any one of SEQ ID NOs: 514-1026 or 1540-2052, or any one of SEQ ID NOs: 514-1026, 1540-2052, 2119-2182, or 2247-2335.

[0013] In some embodiments, the ASO comprises a nucleic acid sequence comprising at least 14 contiguous nucleotides that differ by no more than 1, 2, 3 or 4 nucleotides from any one of SEQ ID NOs: 514-1026 and 1540-2052, or any one of SEQ ID NOs: 514-1026, 1540-2052, 2119-Attorney Docket No. 63578-7176012182, or 2247-2335. In some embodiments, the oligonucleotide comprises at least one or more modifications. In some embodiments, the at least one or more modifications is selected from a ribose modification, a backbone modification, or a nucleobase modification. In some embodiments, the ribose modification comprises a locked nucleic acid (LNA), a tricyclo-DNA (tcDNA), 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-methoxyethyl (2'-M0E), 2'-deoxy-2'-arabino- fluoro, 2'-O-benzyl, 2'-O-(methyl-4-pyridine), 2' cyclic ethyl (cET), phosphorodiamidate morpholino (PMO), glycol nucleic acid (GNA), or unlocked nucleic acid (UNA).

[0014] In some embodiments, the ribose modification comprises a 2'-deoxy-2'-fluoro, 2'-O- methyl, glycol nucleic acid (GNA), unlocked nucleic acid (UNA), or a threose nucleic acid (TNA). In some embodiments, the ribose modification is a 2'-deoxy-2'-fluoro or 2'-O-methyl modification. In some embodiments, the backbone modification comprises phosphorothioate, phosphorodithioate, methylphosphonate, methyoxypropyl-phosphonate, 5'-(E)- vinylphosphonate, 5'-methyl phosphate, 5'-phosphorothioate, or peptide nucleic acid (PNA). In some embodiments, the backbone modification comprises a phosphorothioate. In some embodiments, the backbone modification comprises a phosphorothioate modification.

[0015] In some embodiments, the nucleobase modification comprises 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, 5-methylcytosine (5-Me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, N6-alkyl derivatives, N2- alkyl, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyl uracil, 5- propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxy, 5-halo, 5-trifluoromethyl, N7- methylguanine, N7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7- deazaadenine, 3 -deazaguanine, 3 -deazaadenine, or any combination thereof.

[0016] In some embodiments, the oligonucleotide comprises at least one modified nucleotide selected from the group consisting of a deoxy nucleotide, a 3 ’-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'- deoxy-modified nucleotide, a 2’-5’-linked ribonucleotide (3’-RNA), a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2’-amino-modified nucleotide, a 2’-O-allyl-modified nucleotide, 2’-C-alkyl- modified nucleotide, 2’-hydroxyl-modified nucleotide, a 2’-O-(methoxy ethyl) modified nucleotide, a 2’-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate morpholino, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a nucleotide comprising a 5'-methylphosphonate group, a nucleotide comprising a 5’ phosphate or 5’ phosphate mimic, a nucleotide comprising vinylAttorney Docket No. 63578-717601 phosphonate, a glycol nucleic acid (GNA), a glycol nucleic acid S-Isomer (S-GNA), a nucleotide comprising 2-hydroxymethyl-tetrahydrofurane-5-phosphate, a nucleotide comprising 2’- deoxythymidine-3 ’phosphate, a nucleotide comprising 2 ’-deoxyguanosine-3’ -phosphate; a cytidine-2'-phosphate, a guanosine-2'-phosphate, a uridine-2'-phosphate, an adenosine-2'- phosphate, a 2'-O-hexadecyl-adenosine-3 '-phosphate, a 2'-O-hexadecyl-cytidine-3 '-phosphate, a 2'-O-hexadecyl-guanosine-3'-phosphate, and a 2'-O-hexadecyl-uridine-3'-phosphate, a 3'-3' inverted nucleotide linkage, a 5 ’-5’ inverted nucleotide linkage, TNA and combinations thereof.

[0017] In some embodiments, the nucleotide modification is a deoxy nucleotide, a 3 ’-terminal deoxythymine (dT) nucleotide, a 3 '-3' inverted nucleotide linkage, a 5 ’-5’ inverted nucleotide linkage, or a 5'-(E)-vinylphosphonate-2’-O-methyluridine-3’-phosphate. In some embodiments, the nucleotide modification is 5'-(E)-vinylphosphonate-2’-O-methyluridine-3’-phosphate. In some embodiments, at least one of the modifications is a thermally destabilizing nucleotide modification.

[0018] In some embodiments, the thermally destabilizing nucleotide modification is selected from the group consisting of an abasic modification; a mismatch with the opposing nucleotide in the duplex; and destabilizing sugar modification, a 2’ -deoxy modification, an acyclic nucleotide, an unlocked nucleic acids (UNA), and a glycerol nucleic acid (GNA). In some embodiments, the modification comprises a short sequence of 3 ’-terminal deoxythymine nucleotide (dT). In some embodiments, the modifications on the nucleotides are 2’-O-methyl and 2 ’deoxy-2’ -fluoro modifications. In some embodiments, the oligonucleotide comprises at least one phosphorothioate internucleoside or phosphorodithioate internucleoside linkage. In some embodiments, the oligonucleotide comprises 6-8 phosphorothioate internucleoside linkages. In some embodiments, the oligonucleotide comprises at least 1 phosphorothioate internucleoside linkage at a 5’ end of the sense strand.

[0019] In some embodiments, the oligonucleotide comprises at least 2 phosphorothioate internucleoside linkage at a 5’ end of the sense strand. In some embodiments, the oligonucleotide comprises at least 1 phosphorothioate intemucleoside linkage at a 3’ end of the sense strand. In some embodiments, the oligonucleotide comprises at least 1 phosphorothioate internucleoside linkage at a 5’ end of the antisense strand. In some embodiments, the oligonucleotide comprises at least 2 phosphorothioate intemucleoside linkage at a 5’ end of the antisense strand. In some embodiments, the oligonucleotide comprises at least 1 phosphorothioate intemucleoside linkage at a 3’ end of the sense antisense. In some embodiments, no more than five of the nucleotides of the antisense strand are unmodified nucleotides. In some embodiments, all the nucleotides of the antisense strand are modified oligonucleotides. In some embodiments, no more than five of theAttorney Docket No. 63578-717601 sense strand nucleotides are unmodified nucleotides. In some embodiments, all the nucleotides of the sense strand are modified nucleotides.

[0020] In some embodiments, the antisense strand comprises a chemical modification pattern according to (Nfs)a(nNf)b(ns)cn, wherein: n is a 2'-O-methyl-nucleoside-3’ -phosphate; Nfs is a 2'- deoxy-2'-fluoro-nucleoside-3'-phosphorothioate; Nf is a 2'-deoxy-2'-fluoro-nucleoside-3'- phosphate; and ns is a 2'-O-methyl-nucleoside-3'-phosphorothioate, and wherein a is at least 1, b is at least 5-10, and c is at least 1.

[0021] In some embodiments, the antisense strand comprises the chemical modification pattern NfsNfsnNfnNfnNfnNfnNfnNfnNfnNfnsnsn.

[0022] In some embodiments, the sense strand comprises a chemical modification pattern according to (ns)d(Nfn)eNf, wherein: n is a 2’ -O-methyl-nucleoside-3’ -phosphate; Nf is a 2’- deoxy-2'-fluoro-nucleoside-3’ -phosphate; and ns is a 2’ -O-methyl-nucleoside-3 ’- phosphorothioate, and wherein d is at least 1 and e is at least 5-10.

[0023] In some embodiments, the sense strand comprises the chemical modification pattern nsnsNfnNfnNfnNfnNfnNfnNfnNfnNf. In some embodiments, each of the antisense and the sense strand is 17-23 nucleotides in length, wherein the antisense strand comprises the motif F(SF)nSnn, wherein n is from 2 to about 20, nn is 0 or 1, one of F and S is a 2'-deoxy-2’-fluoro modified nucleoside and the other of F and S is a 2'-O-methyl modified nucleoside. In some embodiments, the antisense strand comprises the chemical modification pattern of nNfnnnNfnNfNfnnnnNfnNfnnnnnnn and the sense strand comprises the chemical modification nnnnnnNfnNfNfNfnnnnnnnnnn, wherein n is a 2’-O-methyl-nucleoside and Nf is a 2’-deoxy-2’- fluoro-nucleoside.

[0024] In some embodiments, each of the antisense and the sense strand is 20-23 nucleotides in length, wherein the antisense strand comprises a region having the formula X1-Y-X2, wherein Y is a region of from about 5 to about 12 linked nucleosides and each of XI and X2 is, independently, a plurality of linked nucleosides having the formula FSFS, where one of F and S is a 2 '-deoxy-2’ -fluoro modified nucleoside and the other of F and S is a 2'-O-methyl modified nucleoside; and each intemucleoside linkage of said first and said second oligomeric compound is, independently, a phosphodiester or a phosphorothioate internucleoside linkage.

[0025] In some embodiments, each of the antisense and the sense strand is 17-23 nucleotides in length, wherein the antisense strand comprises a contiguous sequence of linked nucleosides that define an alternating motif of the formula:

[0026] 5 '-Q(-L-Z-L-Q)n(-L-Z)nn-3' wherein: each L is an internucleoside linking group; either each Q is a 2'-deoxy-2’-fluoro-nucleoside and each Z is a 2'-O-methyl nucleoside; or each Q is aAttorney Docket No. 63578-7176012'-0-methyl nucleoside and each Z is a 2'-deoxy-2’-fluoro nucleoside; and n is from 8 to 14 and nn is 0 or 1.

[0027] In some embodiments, the antisense strand is 19-25 nucleotides in length and is represented by the formula:wherein: Bl', B2', B3', and B4' each independently represent a nucleotide containing a modification selected from the group consisting of 2'-O-alkyl, 2 '-substituted alkoxy, 2'- substituted alkyl, 2'-halo, ENA, and BNA / LNA; T1 ', T2', and T3' each independently represent a nucleotide comprising a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-deoxy-2’ -fluoro, and 5 '-methyl-2'-deoxy-2’ -fluoro nucleotides; ql is independently 4 to 15 nucleotides in length; q3 or q7 is independently 1-6 nucleotide(s) in length; q2 or q6 is independently 1-3 nucleotide(s) in length; q4 is independently 0-3 nucleotide(s) in length; and q5 is independently 0-10 nucleotide(s) in length; and wherein: the antisense strand has 2 '-deoxy-2’ -fluoro modifications, and wherein the 2 '-deoxy-2’ -fluoro modifications on the antisense strand consist of four, and only four, 2 '-deoxy-2 ’-fluoro modifications or six, and only six, 2 '-deoxy-2 ’-fluoro modifications.

[0028] In some embodiments, the antisense strand and sense strand are each 14 to 40 nucleotides, and is represented by the formula:wherein: Bl, Bl', B2', B3', and B4' each independently represent a nucleotide containing a modification selected from the group consisting of 2'-O-alkyl, 2 '-substituted alkoxy, 2'- substituted alkyl, 2'-halo, ENA, and BNA / LNA; Cl is a thermally destabilizing nucleotide, selected from the group consisting of i) a nucleotide that forms a mismatch pair with theAttorney Docket No. 63578-717601 opposing nucleotide in the antisense strand, ii) a nucleotide having an abasic modification, and iii) a nucleotide having a sugar modification, and placed at a site opposite to the seed region (positions 2-8) of the antisense strand; T1 ', T2', and T3' each independently represent a nucleotide comprising a modification providing the nucleotide a steric bulk that is less than or equal to the steric bulk of a 2'-0Me modification, wherein the modification is at the 2'-position of a ribose sugar of the nucleotide or at a position of a non-ribose nucleotide similar to the 2'- position of a ribose sugar; each nl, and ql is independently 4 to 15 nucleotides in length; each q3, and q7 is independently 1-6 nucleotide(s) in length; each q2 and q6 is independently 1-3 nucleotide(s) in length; q5 is independently 0-10 nucleotide(s) in length; each n4, and q4 is independently 0-3 nucleotide(s) in length; n2 is 3 nucleotides in length, and T1 each are 2'- deoxy-2’ -fluoro nucleotides; n3 is 7 nucleotides in length, and B2 each are 2'-0Me nucleotides; and n5 is 3 nucleotides in length, and B3 each are 2'-0Me nucleotides.

[0029] In some embodiments, the antisense strand and sense strand are each 19-25 nucleotides in length, wherein the sense strand is represented by the formula:(Is)wherein: Bl, B2, and B3 each independently represent a nucleotide containing a modification selected from the group consisting of 2'-O-alkyl, 2 '-substituted alkoxy, 2 '-substituted alkyl, 2'- halo, ENA, and BNA / LNA; Cl is a thermally destabilizing nucleotide, selected from the group consisting of i) a nucleotide that forms a mismatch pair with the opposing nucleotide in the antisense strand, ii) a nucleotide having an abasic modification, and iii) a nucleotide having a sugar modification, and placed at a site opposite to the seed region (positions 2-8) of the antisense strand; T1 represents a nucleotide comprising a 2 '-deoxy-2’ -fluoro modification; nl or n3 is independently 4 to 15 nucleotides in length; n5 is 1-6 nucleotide(s) in length; n2 is 3; n4 is 0-3 nucleotide(s) in length; and wherein the sense strand has 2 '-deoxy-2 ’-fluoro modifications, and wherein the 2'-deoxy-2’-fluoro modifications on the sense strand consist of four, and only four, 2'-deoxy-2’ -fluoro modifications, wherein the four 2 '-deoxy -2 ’-fluoro modifications are at positions 7 and 9-11 from the 5 '-end of the sense strand.

[0030] In some embodiments, the antisense strand is complementary to at least one portion of a mRNA of the target gene, wherein: the sense strand has 19-22 nucleotides, the antisense strand has 19-25 nucleotides; and the oligonucleotide is represented by the formula:Attorney Docket No. 63578-717601(I)wherein: Bl', B2', B3', and B4' each independently represent a nucleotide containing a modification selected from the group consisting of 2'-O-methyl and 2 '-deoxy-2’ -fluoro; each Bl, B2, and B3 is 2'-O-methyl nucleotide; Cl is glycerol nucleic acid (GNA) placed at a site opposite to the seed region (positions 2-8) of the antisense strand; T1 ', T2', and T3' are each 2'-F, wherein: T1 ' is at position 14 from the 5' end of the antisense strand, and q2 is 1; and T3' is at position 2 from the 5' end of the antisense strand, and q6 and q7 are 1; each nl, n3, and ql is independently 4 to 15 nucleotides in length; each n5 and q3 is independently 1-6 nucleotide(s) in length; q5 is independently 0-10 nucleotide(s) in length; each n4 and q4 is independently 0-3 nucleotide(s) in length; n2 is 3 nucleotides in length, and T1 each are 2 '-deoxy-2 ’-fluoro nucleotides, and wherein the oligonucleotide is covalently conjugated to at least one ligand; and one of the T1 nucleotides is at position 11 from the 5' end of the sense strand.

[0031] In some embodiments, the sense strand has 19-22 nucleotides, the antisense strand has 19-25 nucleotides; and the oligonucleotide is represented by the formula:(i)wherein: Bl', B2', B3', and B4' each independently represent a nucleotide containing a modification selected from the group consisting of 2'-O-methyl and 2 '-deoxy-2 ’-fluoro; each Bl, B2, and B3 is 2'-0Me; Cl is glycerol nucleic acid (GNA) placed at a site opposite to the seed region (positions 2-8) of the antisense strand; T1 ', T2', and T3' are each 2 '-deoxy-2 ’-fluoro, wherein: T1 ' is at position 14 from the 5' end of the antisense strand, and q2 is 1; and T3' is at position 2 from the 5' end of the antisense strand, and q6 and q7 are 1; each nl, n3, and ql is independently 4 to 15 nucleotides in length; each n5 and q3 is independently 1-6 nucleotide(s) in length; q5 is independently 0-10 nucleotide(s) in length; each n4 and q4 is independently 0-3 nucleotide(s) in length; n2 is 3 nucleotides in length, and T1 each are 2 '-deoxy-2 ’-fluoro, andAttorney Docket No. 63578-717601 wherein, the oligonucleotide is covalently conjugated to at least one ligand; and one of the T1 nucleotides is at a position in the sense strand that is opposite to position 11 from the 5' end of the antisense strand; and the oligonucleotide comprises at least one phosphorothioate internucleoside linkage.

[0032] In some embodiments, the antisense strand and sense strand are each 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to a target sequence to mediate RNA interference, wherein said sense strand comprises at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5' region of the antisense strand or a precursor thereof, wherein the antisense strand further comprises one or both of the following characteristics: 2, 3, 4, 5 or 6 2 '-deoxy-2’ -fluoro modifications; and (ii) 1, 2, 3, 4 or 5 phosphorothioate internucleoside linkages; and said sense strand comprises one, two or three of the following characteristics: (iii) 2, 3, 4, or 5 2 '-deoxy-2 ’-fluoro modifications; and (iv) 1, 2, 3, 4 or 5 phosphorothioate intemucleoside linkages.

[0033] In some embodiments, the antisense strand comprises a nucleotide sequence according to any one of SEQ ID NOs: 514-1026, or any one of SEQ ID NOs: 514-1026 or 2119-2182. In some embodiments, the sense strand comprises a nucleotide sequence according to any one of SEQ ID NOs: 1-513, or any one of SEQ ID NOs: 1-513 or 2055-2118. In some embodiments, the administration of the oligonucleotide results in at least a 55% reduction in expression levels of an GLIS2 mRNA in kidney cells. In some embodiments, the administration of the oligonucleotide results in at least a 75% reduction in expression levels of an GLIS2 mRNA in kidney cells. In some embodiments, the administration of the oligonucleotide results in at least an 80% reduction in expression levels of an GLIS2 mRNA in kidney cells.

[0034] In some embodiments, the administration of the oligonucleotide results in at least an 85% reduction in expression levels of an GLIS2 mRNA in kidney cells. In some embodiments, the oligonucleotide further comprises a terminal, chiral modification occurring at the first internucleoside linkage at the 3 ’-end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first intemucleoside linkage at the 5 ’-end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first intemucleoside linkage at the 5 ’-end of the sense strand, having the linkage phosphorus atom in either Rp configuration or Sp configuration.

[0035] In some embodiments, the oligonucleotide further comprises a terminal, chiral modification occurring at the first and second intemucleoside linkages at the 3 ’-end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first intemucleoside linkage at the 5 ’-end of the antisense strand,Attorney Docket No. 63578-717601 having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleoside linkage at the 5 ’-end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

[0036] In some embodiments, the oligonucleotide further comprises a terminal, chiral modification occurring at the first, second and third internucleoside linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first intemucleoside linkage at the 5 ’-end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleoside linkage at the 5 ’-end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

[0037] In some embodiments, the oligonucleotide further comprises a terminal, chiral modification occurring at the first, and second internucleoside linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the third intemucleoside linkages at the 3 ’-end of the antisense strand, having the linkage phosphorus atom in Rp configuration, a terminal, chiral modification occurring at the first intemucleoside linkage at the 5’-end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first intemucleoside linkage at the 5 ’-end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

[0038] In some embodiments, the oligonucleotide further comprises a terminal, chiral modification occurring at the first, and second intemucleoside linkages at the 3 ’-end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first, and second intemucleoside linkages at the 5 ’-end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first intemucleoside linkage at the 5 ’-end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration. In some embodiments, the oligonucleotide further comprises a phosphate or phosphate mimic at the 5’-end of the antisense strand. In some embodiments, the phosphate mimic is a 5 ’-vinyl phosphonate (VP).

[0039] In one aspect, provided herein is a pharmaceutical composition comprising the oligonucleotide of any one of aspects and embodiments herein.

[0040] In some embodiments, the pharmaceutical composition further comprises a targeting moiety. In some embodiments, the targeting moiety is a targeting ligand. In some embodiments, the targeting ligand is a small molecule-based, sugar-based, fatty acid-based, protein-based, or nucleic acid-based targeting ligand. In some embodiments, the protein-based targeting ligand is an antibody, nanobody, affibody, peptibody, or a peptide.Attorney Docket No. 63578-717601

[0041] In one aspect, provided herein is a method for inhibiting GLIS2 expression in a subject, the method comprising administering an effective amount of the oligonucleotide of any one of aspects or embodiments herein or the pharmaceutical composition of any one of aspects or embodiments herein to the subject. In some embodiments, the subject is a human. In some embodiments, the reduction of GLIS2 mRNA or protein expression levels is measured in a population of kidney cells derived from the subject.

[0042] In some embodiments, the reduction of GLIS2 mRNA or protein expression levels is measured in tissues derived from the subject. In some embodiments, the method further comprises administering to the subject an additional agent or a therapy suitable for treatment or prevention of an GLIS2 related disorder.DETAILED DESCRIPTION

[0043] Provided herein are oligonucleotides for inhibiting the expression of GLIS2. In some embodiments, the oligonucleotide comprises an antisense strand substantially complementary to a sequence encoding GLIS2. In some embodiments, the antisense strand comprises at least 14 contiguous nucleotides substantially complementary to the sequence of nucleotides encoding GLIS2, with no more than 4 mismatched nucleotides.

[0044] The term “antisense strand” refers to an oligonucleotide having a nucleotide sequence substantially complementary to a target sequence in a transcript, e.g., an mRNA encoding GLIS2. In embodiments wherein the oligonucleotide is an RNAi agent, the term antisense strand may be used interchangeably with the term “guide strand”.

[0045] The term “sense strand” refers to an oligonucleotide having a nucleotide sequence substantially complementary to an antisense strand, e.g., the antisense strand of an RNAi agent herein. The term “sense strand” may be used interchangeably with the term “passenger strand.”

[0046] As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide comprising the second nucleotide sequence, as will be understood by the skilled person.

[0047] The terms “complementary,” “completely complementary” and “substantially complementary” herein can be used with respect to the base matching between the antisense strand of an oligonucleotide, e.g., an RNAi agent, and a target sequence, or between the sense strand and the antisense strand of an RNAi agent, as will be understood from the context of their use. Complementary sequences, e.g., between an antisense strand and a target sequence in a target transcript, or between the sense and antisense strand of an siRNA, include base-pairing ofAttorney Docket No. 63578-717601 the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “completely complementary” with respect to each other when there are 0 mismatched base pairs upon hybridization of the two sequences. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be completely complementary, or they can form one or more, but generally not more than 5, 4, 3, 2, or 1 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RNA-induced silencing complex (RISC) pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, an siRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 19 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 19 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “completely complementary” for the purposes described herein.

[0048] “Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogsteen base pairing.

[0049] If an RNA sequence is recited using deoxyribonucleotides, any thymidines (“T”s) can be replaced with uridines (“U”s) or uridine analogs to convert the DNA sequence to an RNA sequence.

[0050] In some embodiments, the oligonucleotide comprises an antisense strand comprising at least 14 contiguous nucleotides substantially complementary to a sequence of nucleotides within a transcript encoding GLIS2. In some embodiments, the antisense strand has no more than 5 mismatched nucleotides to the sequence of nucleotides within the transcript encoding GLIS2. In some embodiments, the antisense strand has no more than 4 mismatched nucleotides to the sequence of nucleotides within the transcript encoding GLIS2. In some embodiments, the antisense strand has no more than 3 mismatched nucleotides to the sequence of nucleotides within the transcript encoding GLIS2. In some embodiments, the antisense strand has no more than 2 mismatched nucleotides to the sequence of nucleotides within the transcript encoding GLIS2. In some embodiments, the antisense strand has no more than 1 mismatched nucleotidesAttorney Docket No. 63578-717601 to the sequence of nucleotides within the transcript encoding GLIS2. In some embodiments, the antisense strand is completely complementary to the sequence of nucleotides within the transcript encoding GLIS2 (e.g., 0 mismatches).

[0051] GLIS2 encodes one of three Gli-similar (Glisl-3) Kriippel-like zinc finger transcription factor proteins. GLIS2 is most abundantly expressed in the kidney along the entire nephron and is the causative gene for nephronophthisis type 7 (NPHP7). The protein encoded by GLIS2 is a transcription factor comprising a single protein. Exemplary sequences of GLIS2 may be found for example at NCBI RefSeq ID NM 001318918.2 (SEQ ID NO: 2053). In some embodiments, the GLIS2 comprises a nucleotide sequence corresponding to the transcript having the NCBI RefSeq ID NM_032575.3 (SEQ ID NO: 2054).

[0052] In some embodiments, the sequence of nucleotides encoding GLIS2 comprises a nucleotide sequence having at least 75% nucleotide sequence identity to any one of SEQ ID NOs: 2053-2054. In some embodiments, the sequence of nucleotides encoding GLIS2 comprises a nucleotide sequence having at least 80% nucleotide sequence identity to any one of SEQ ID NOs: 2053-2054. In some embodiments, the sequence of nucleotides encoding GLIS2 comprises a nucleotide sequence having at least 85% nucleotide sequence identity to any one of SEQ ID NOs: 2053-2054. In some embodiments, the sequence of nucleotides encoding GLIS2 comprises a nucleotide sequence having at least 90% nucleotide sequence identity to any one of SEQ ID NOs: 2053-2054. In some embodiments, the sequence of nucleotides encoding GLIS2 comprises a nucleotide sequence having at least 95% nucleotide sequence identity to any one of SEQ ID NOs: 2053-2054. In some embodiments, the sequence of nucleotides encoding GLIS2comprises a nucleotide sequence having at least 99% nucleotide sequence identity to any one of SEQ ID NOs: 2053-2054.

[0053] In some embodiments, the sequence of nucleotides encoding GLIS2 comprises a nucleotide sequence having at least 75% nucleotide sequence identity to SEQ ID NO: 2053. In some embodiments, the sequence of nucleotides encoding GLIS2 comprises a nucleotide sequence having at least 80% nucleotide sequence identity to SEQ ID NO: 2053. In some embodiments, the sequence of nucleotides encoding GLIS2 comprises a nucleotide sequence having at least 85% nucleotide sequence identity to SEQ ID NO: 2053. In some embodiments, the sequence of nucleotides encoding GLIS2 comprises a nucleotide sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 2053. In some embodiments, the sequence of nucleotides encoding GLIS2 comprises a nucleotide sequence having at least 95% nucleotide sequence identity to SEQ ID NO: 2053. In some embodiments, the sequence of nucleotides encoding GLIS2 comprises a nucleotide sequence having at least 99% nucleotide sequence identity to SEQ ID NO: 2053.Attorney Docket No. 63578-717601

[0054] In some embodiments, the sequence of nucleotides encoding GLIS2 comprises a nucleotide sequence having at least 75% nucleotide sequence identity to SEQ ID NO: 2054. In some embodiments, the sequence of nucleotides encoding GLIS2 comprises a nucleotide sequence having at least 80% nucleotide sequence identity to SEQ ID NO: 2054. In some embodiments, the sequence of nucleotides encoding GLIS2 comprises a nucleotide sequence having at least 85% nucleotide sequence identity to SEQ ID NO: 2054. In some embodiments, the sequence of nucleotides encoding GLIS2 comprises a nucleotide sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 2054. In some embodiments, the sequence of nucleotides encoding GLIS2 comprises a nucleotide sequence having at least 95% nucleotide sequence identity to SEQ ID NO: 2054. In some embodiments, the sequence of nucleotides encoding GLIS2 comprises a nucleotide sequence having at least 99% nucleotide sequence identity to SEQ ID NO: 2054. In some embodiments, the antisense strand is substantially complementary to a sequence of nucleotides corresponding to an untranslated region of the GLIS2 transcript. In some embodiments, the antisense strand is substantially complementary to the sequence of nucleotides corresponding to an untranslated region of the GLIS2 transcript with no more than 5, 4, 3, 2, 1, or 0 mismatches. In some embodiments, the antisense strand is completely complementary to the sequence of nucleotides corresponding to an untranslated region of the GLIS2 transcript.RNAi Agents

[0055] In some embodiments, the oligonucleotide is an RNAi agent or RNAi trigger for inhibiting expression of GLIS2. As used herein, an “RNAi agent” or “RNAi trigger” refers to an oligonucleotide molecule capable of inducing RNA interference (RNAi), which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. The RNAi agents disclosed herein include but are not limited to: short (or small) interfering RNAs (siRNAs), double stranded RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), and dicer substrates e.g., DsiRNAs).

[0056] In some embodiments, the RNAi agent is a double stranded RNA molecule comprising an antisense strand and a sense strand that are complementary to one another and hybridize to form a duplex or double stranded region. One strand of the RNAi agent, the antisense strand or guide strand, includes a region of complementarity to a target sequence in GLIS2. The other strand, the sense strand or passenger strand, includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. In some embodiments, the double stranded RNA molecule may be formed by base pairing between two separate molecules of RNA (e.g., an antisense strand and aAttorney Docket No. 63578-717601 sense strand). In some embodiments, the double stranded RNA molecule is a self- complementary molecule formed by intramolecular base pairing between two separate regions of a single RNA molecule (e.g., an antisense region linked to a sense strand through an unpaired RNA linker forming a loop or hairpin loop).

[0057] Where the two strands are part of a self-complementary molecule, the connecting RNA chain is referred to as a “hairpin loop”. A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides or nucleotides not directed to the target site of the dsRNA. In some embodiments, the hairpin loop can be 10 or fewer nucleotides. In some embodiments, the hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, the hairpin loop can be 4-8 nucleotides. In some embodiments, the hairpin loop can contain 1-4 oligoethyne glycols, including ethylene glycol, di ethylene glycol, triethylene glycol, tetraethylene glycol, hexaethylene glycol, or any combination thereof.

[0058] Where the two substantially complementary strands of a double stranded RNA molecule comprise separate RNA molecules, those molecules need not, but can be covalently connected. In certain embodiments, where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3 ’-end of one strand and the 5 ’-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker” (though it is noted that certain other structures defined elsewhere herein can also be referred to as a “linker”). The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the double stranded RNA molecule minus any overhangs that are present in the duplex.

[0059] In some embodiments, the RNAi agent is an siRNA.

[0060] In some embodiments, the RNAi agent is a shRNA.

[0061] In some embodiments, the RNAi agent is a dicer substrate (e.g., a Dicer-substrate siRNA).

[0062] In some embodiments, the sense and antisense strands of the dsRNA are each independently about 15 to about 30 nucleotides in length, or about 25 to about 30 nucleotides in length, e.g., each strand is independently between 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15- 23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19- 21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length.Attorney Docket No. 63578-717601

[0063] In some embodiments, the duplex structure is between 15 and 30 base pairs in length, e.g, between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15- 18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20- 27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21- 24, 21-23, or 21-22 base pairs in length.

[0064] An RNAi agent as described herein can further include one or more single-stranded nucleotide overhangs, e.g., an overhang of 1, 2, 3, or 4 nucleotides. RNAi agent having at least one nucleotide overhang can have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. A nucleotide overhang can comprise or consist of a nucleotide / nucleoside analog, including a deoxynucleotide, an inverted deoxynucleotide or an inverted abasic nucleotide. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5'- end, 3'-end or both ends of either an antisense or sense strand of the RNAi agent. In certain embodiments, longer, extended overhangs are possible.

[0065] In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 19 nucleotides in length.

[0066] The oligonucleotides disclosed herein may be unmodified or modified (e.g., chemically modified or conjugated). A modified oligonucleotide as disclosed herein comprises an identical nucleobase sequence as compared to a corresponding unmodified oligonucleotide, but further comprises one or more modifications as disclosed herein.

[0067] In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides differing by no more than 4 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 514-1026 or 1540-2052, or any one of SEQ ID NOs: 514- 1026, 1540-2052, 2119-2182, or 2247-2335. In some embodiments, the antisense strand differs by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 514- 1026, or any one of SEQ ID NOs: 514-1026, 1540-2052, 2119-2182, or 2247-2335. In some embodiments, the antisense strand differs by no more than 2 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 514-1026 or 1540-2052, or any one of SEQ ID NOs: 514- 1026, 1540-2052, 2119-2182, or 2247-2335. In some embodiments, the antisense strand differs by no more than 1 nucleotide from the nucleotide sequence of any one of SEQ ID NOs: 514- 1026 or 1540-2052, or any one of SEQ ID NOs: 514-1026, 1540-2052, 2119-2182, or 2247- 2335. In some embodiments, the antisense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 514-1026 or 1540-2052, or any one of SEQ ID NOs: 514-1026, 1540-2052, 2119-2182, or 2247-2335. In some embodiments, the antisense strand consists of the nucleotideAttorney Docket No. 63578-717601 sequence of any one of SEQ ID NOs: 514-1026 or 1540-2052, or any one of SEQ ID NOs: 514- 1026, 1540-2052, 2119-2182, or 2247-2335.

[0068] In some embodiments, the antisense strand comprises a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of any one of SEQ ID NOs: 514-1026 or 1540-2052, or any one of SEQ ID NOs: 514-1026, 1540-2052, 2119- 2182, or 2247-2335.

[0069] In some embodiments, the antisense strand consists of a nucleotide sequence having the nucleotide sequence of any one of SEQ ID NOs: 514-1026 or 1540-2052, or any one of SEQ ID NOs: 514-1026, 1540-2052, 2119-2182, or 2247-2335.

[0070] In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides differing by no more than 4 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 1-513 or 1027-1539, or any one of SEQ ID NOs: 1-513, 1027-1539, 2055-2118, or 2183-2246. In some embodiments, the sense strand differs by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 1-513, or any one of SEQ ID NOs: 1-513, 1027-1539, 2055-2118, or 2183-2246. In some embodiments, the sense strand differs by no more than 2 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 1-513 or 1027-1539, or any one of SEQ ID NOs: 1-513, 1027-1539, 2055-2118, or 2183- 2246. In some embodiments, the sense strand differs by no more than 1 nucleotide from the nucleotide sequence of any one of SEQ ID NOs: 1-513 or 1027-1539, or any one of SEQ ID NOs: 1-513, 1027-1539, 2055-2118, or 2183-2246. In some embodiments, the sense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 1-513 or 1027-1539, or any one of SEQ ID NOs: 1-513, 1027-1539, 2055-2118, or 2183-2246. In some embodiments, the sense strand consists of the nucleotide sequence of any one of SEQ ID NOs: 1-513 or 1027-1539, or any one of SEQ ID NOs: 1-513, 1027-1539, 2055-2118, or 2183-2246.

[0071] In some embodiments, the sense strand comprises a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of any one of SEQ ID NOs: 1-513 or 1027-1539, or any one of SEQ ID NOs: 1-513, 1027-1539, 2055-2118, or 2183- 2246.

[0072] In some embodiments, the sense strand consists of a nucleotide sequence having the nucleotide sequence of any one of SEQ ID NOs: 1-513 or 1027-1539, or any one of SEQ ID NOs: 1-513, 1027-1539, 2055-2118, or 2183-2246.

[0073] In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides differing by no more than 4 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 514-1026 or 1540-2052, or any one of SEQ ID NOs: 514- 1026, 1540-2052, 2119-2182, or 2247-2335, and the sense strand comprises a nucleotideAttorney Docket No. 63578-717601 sequence at least 15 contiguous nucleotides differing by no more than 4 nucleotides from the nucleotide sequence of any one of SEQ ID Nos: 1-513 or 1027-1539, or any one of SEQ ID NOs: 1-513, 1027-1539, 2055-2118, or 2183-2246.

[0074] In some embodiments, the antisense strand comprises a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of any one of SEQ ID NOs: 514-1026 or 1540-2052, or any one of SEQ ID NOs: 514-1026, 1540-2052, 2119- 2182, or 2247-2335, and the sense strand comprises a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of any one of SEQ ID NOs: 1-513 or 1027-1539, or any one of SEQ ID NOs: 1-513, 1027-1539, 2055-2118, or 2183-2246.

[0075] In some embodiments, the antisense strand consists of a nucleotide sequence of any one of SEQ ID NOs: 514-1026 or 1540-2052, or any one of SEQ ID NOs: 514-1026, 1540-2052, 2119-2182, or 2247-2335, and the sense strand consists of a nucleotide sequence of any one of SEQ ID Nos: 1-513 or 1027-1539, or any one of SEQ ID NOs: 1-513, 1027-1539, 2055-2118, or 2183-2246.

[0076] While a target sequence is generally about 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and knowledge within the art provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an RNAi agent, mediate the best inhibition of target gene expression (e.g., GLIS2. Thus, while the sequences described herein represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

[0077] It is contemplated that for any sequence identified, e.g., any one of SEQ ID NOs: 1-2052, or any one of SEQ ID NOs: 1-2052 or 2055-2335, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences andAttorney Docket No. 63578-717601 testing those sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of RNAi agents based on those target sequences in an inhibition assay as known in the art and / or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and / or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor.

[0078] An GLIS2 RNAi agent described herein can contain one or more mismatches to the target sequence. In some embodiment, an GLIS2 RNAi agent as described herein contains no more than 4 mismatches (e.g., no more than 4, 3, 2, 1, or 0 mismatches to the target sequence). If the antisense strand of the RNAi agent contains mismatches to a target sequence, it may be preferable that the area of mismatch is not located in the center of the region of complementarity. If the antisense strand of the RNAi agent contains mismatches to the target sequence, it may be preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5’ - or 3’-end of the region of complementarity. For example, for a 21 nucleotide RNAi agent, the strand which is complementary to a region of, e.g., GLIS2, generally does not contain any mismatch within the central 11 nucleotides. The methods described herein or methods known in the art can be used to determine whether an RNAi agent containing a mismatch to a target sequence is effective in inhibiting the expression of GLIS2. Consideration of the efficacy of RNAi agents with mismatches in inhibiting expression of a target gene is important, especially if the particular region of complementarity in a target gene is known to have polymorphic sequence variation within the population.

[0079] In some embodiments, any one of the RNAi agents described herein can reduce expression levels of GLIS2. In some embodiments, reduction in expression levels is assayed using an GLIS2 reporter construct. The skilled artisan will appreciate that various approaches to generating a reporter construct for assessing RNAi agent activity may be used in the art, including commercially available kits, e.g., the psi CHECK-2 vector (Promega). In some embodiments, any one of the RNAi agents described herein can reduce expression levels of an GLIS2 reporter when assayed in cultured cells. Any suitable cells known in the art may be used to assess the RNAi agents. In some embodiments, the cultured cells are Hepal-6. In some embodiments, administration of any one of the RNAi agents disclosed herein to a cultured cellAttorney Docket No. 63578-717601 results in a reduction in expression level of an GLIS2. In some embodiments, administration of the RNAi agent results in at least a 55% reduction in expression levels of the GLIS2 reporter. In some embodiments, administration of the RNAi agent results in at least a 60% reduction in expression levels of the GLIS2 reporter. In some embodiments, administration of the RNAi agent results in at least a 65% reduction in expression levels of the GLIS2 reporter. In some embodiments, administration of the RNAi agent results in at least a 70% reduction in expression levels of the GLIS2 reporter. In some embodiments, administration of the RNAi agent results in at least a 75% reduction in expression levels of the GLIS2 reporter. In some embodiments, administration of the RNAi agent results in at least an 80% reduction in expression levels of the GLIS2 reporter. In some embodiments, administration of the RNAi agent results in at least an 85% reduction in expression levels of the GLIS2 reporter. In some embodiments, administration of the RNAi agent results in at least a 90% reduction in expression levels of the GLIS2 reporter. In some embodiments, administration of the RNAi agent results in at least a 95% reduction in expression levels of the GLIS2 reporter.Single-stranded antisense oligonucleotides

[0080] In some embodiments, the oligonucleotide is a single-stranded antisense oligonucleotide, or “ASO.” ASOs comprise an antisense strand with at least partial complementary to a target sequence in an RNA. Upon binding to a target sequence, downregulation of the RNA may be achieved through various mechanisms, including, but not limited to, sterically blocking translation or recruitment of RNase H.

[0081] In some embodiments, the ASO comprises a nucleic acid sequence of at least 14 contiguous nucleotides that differ by no more than 1, 2, 3, or 4 nucleotides from any one of SEQ ID NOs: 514-1026 or 1540-2052, or any one of SEQ ID NOs: 514-1026, 1540-2052, 2119-2182, or 2247-2335. In some embodiments, the ASO comprises a nucleotide sequence of any one of SEQ ID NOs: 514-1026 or 1540-2052, or any one of SEQ ID NOs: 514-1026, 1540-2052, 2119- 2182, or 2247-2335.

[0082] In some embodiments, the ASO comprises 2'-deoxy ribonucleotides and phosphorothioate internucleoside linkages.

[0083] In some embodiments, the ASO is a “gapmer” ASO comprising a 2'-deoxy "gap" region flanked by "wings" having nucleotides with 2'-modified ribonucleotides. In some embodiments, the ASO is a "MOE gapmer" in which the 2'-modified ribonucleotide is a 2'-O-methoxyethyl (2'- MOE or simply MOE) modification, and each of the internucleoside linkages is a phosphorothi oate .Attorney Docket No. 63578-717601Modified Nucleotides

[0084] The oligonucleotides disclosed herein may be modified or unmodified. In some embodiments, any one of the oligonucleotides contain one or more modifications. As used herein, a modification to a nucleotide or “modified nucleotide” refers to any nucleotide other than the canonical ribonucleotides adenine, guanine, cytosine, and uracil.

[0085] In some embodiments, the oligonucleotide comprises at least one or more modified nucleotides. In some embodiments, no more than 1, 2, 3, 4, or 5 of the nucleotides of the oligonucleotide are unmodified nucleotides. In some embodiments, all nucleotides of the oligonucleotide are modified nucleotides.

[0086] In some embodiments, wherein the oligonucleotide is an RNAi agent, the antisense strand comprises at least one or more modified nucleotides. In some embodiments, no more than 1, 2, 3, 4, or 5 of the nucleotides of the antisense strand are unmodified nucleotides. In some embodiments, all nucleotides of the antisense strand are modified nucleotides.

[0087] In some embodiments, wherein the oligonucleotide is an RNAi agent, the sense strand comprises at least one or more modified nucleotides. In some embodiments, no more than 1, 2, 3, 4, or 5 of the nucleotides of the sense strand are unmodified nucleotides. In some embodiments, all nucleotides of the sense strand are modified nucleotides.

[0088] Modified nucleotides include, but are not limited to 2'-modified nucleotides, 3' to 3' linkages (inverted) nucleotides, bridged nucleotides, 2',3'-seco nucleotide mimics e.g., unlocked nucleobase analogues (UNAs), locked nucleotides (LNAs), 5'-(S)-methyl-2’-deoxy-2’- fluoronucleotide (5’Me-Nf), vinyl phosphonate deoxyribonucleotides, vinyl phosphonate nucleotides, a nucleotide comprising a 5'-phosphorothioate group, a nucleotide comprising a 5'- methylphosphonate group, a nucleotide comprising a 5’ phosphate or 5’ phosphate mimic and N- (methane sulfonyl) phosphoramidate group. In some embodiments, 2'-modified nucleotides (e.g., a nucleotide with a group other than a hydroxyl group at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides (represented herein as a lower case letter n in a nucleotide sequence), 2'-deoxy-2'-fluoro nucleotides (represented herein as Nf, also represented herein as 2'-fluoro nucleotide),

[0089] In some embodiments, modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 5' to 5' linkages (inverted) nucleotides, non-natural base-comprising nucleotides, peptide nucleic acids (PNAs), 3'-O- methoxy (2' intemucleoside linked) nucleotides, 2’-deoxy-2’-fluoro-arabino nucleotides, cyclopropyl phosphonate nucleotides (cPrpN), vinyl phosphonate 2’ -(methoxy ethyl) unlocked nucleotides, a nucleotide comprising 2-hydroxymethyl-tetrahydrofurane-5-phosphate, 2'-deoxy nucleotides (represented herein as dN), 2'-methoxy ethyloxy (2'-O-(2-methoxylethyl))Attorney Docket No. 63578-717601 nucleotides, 2'-amino nucleotides, and 2'-alkyl nucleotides. It is not necessary for all positions in a given compound to be uniformly modified. Conversely, more than one modification may be incorporated in a single oligonucleotide. Modification at one nucleotide is independent of modification at another nucleotide.

[0090] In some embodiments, the nucleotide modification comprises a deoxyribonucleotide, a 3 ’-terminal deoxythymine (dT) nucleotide, an abasic nucleotide, a 2'-modified nucleotide, a 3' to 3' linkages (inverted) nucleotide, a 5' to 5' linkages (inverted) nucleotide, a non-natural basecomprising nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a nucleotide comprising a 5'-methylphosphonate group, a nucleotide comprising a 5’ phosphate or 5’ phosphate mimic, a nucleotide comprising 2-hydroxymethyl-tetrahydrofurane-5-phosphate, or a nucleotide comprising a -( methane sulfonyl) phosphoramidate group.

[0091] In some embodiments, at least one or more modifications is selected from a ribose modification, a backbone modification, or a nucleobase modification. In some embodiments, the one or more modifications is a combination of a ribose modification, a backbone modification, and / or a nucleobase modification.

[0092] In some embodiments, the oligonucleotide comprises one or more nucleobase modifications. Nucleobase modifications include, for example, synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5- methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2- alkyl e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo e.g., 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine, 7-deazaadenine, 3 -deazaguanine, and 3 -deazaadenine, or combinations thereof.

[0093] In some embodiments, the oligonucleotide comprises one or more backbone modification or non-standard linkages e.g., modified intemucleoside linkages). In some embodiments, a backbone modification is a non-phosphate-containing covalent intemucleoside linkage. Modified internucleoside linkages or backbones include, but are not limited to, 5’-phosphorothioate groups (represented herein as a lower case “s”), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates e.g., methyl phosphonates or 3 '-alkylene phosphonates), chiral phosphonates, phosphinates,Attorney Docket No. 63578-717601 phosphoramidates (e.g., 3'-amino phosphoramidate, aminoalkylphosphoramidates, or thionophosphoramidates), thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some embodiments, a modified backbone or modified intemucleoside linkage lacks a phosphorus atom. Modified internucleoside linkages lacking a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter-sugar linkages. In some embodiments, modified intemucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methylene formacetyl and thioform acetyl backbones, alkene- containing backbones, sulfamate backbones, methyieneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH2 components.

[0094] In some embodiments, the backbone modification comprises phosphorothioate.

[0095] In some embodiments, any one of the oligonucleotides disclosed herein further comprise a sequence of 3 ’-terminal deoxythymine nucleotides (dTs). In some embodiments, any one of the antisense strands described herein further comprises at least one terminal dT. In some embodiments, any one of the antisense strands described herein further comprises 2 or more terminal dTs, e.g., 2, 3, 4, or more terminal dTs. In some embodiments, wherein the oligonucleotide is an RNAi agent, any one of the sense strands described herein further comprises at least one terminal dT. In some embodiments, any one of the sense strands described herein further comprises 2 or more terminal dTs, e.g., 2, 3, 4, or more terminal dTs. In some embodiments, the antisense and sense strand of an RNAi agent herein each contain one or more terminal dTs.

[0096] In some embodiments, any one of the oligonucleotides disclosed herein further comprises at least one phosphorothioate intemucleoside or phosphorodithioate intemucleoside linkage. In some embodiments, any one of the oligonucleotides disclosed herein further comprises at least one phosphorothioate intemucleoside linkage. In some embodiments, at least one phosphorothioate intemucleoside linkage is at the 5’ end of the antisense strand. In some embodiments, the oligonucleotide comprises at least 2 phosphorothioate intemucleoside linkages at the 5’ end of the antisense strand. In some embodiments, at least one phosphorothioate intemucleoside linkage is at the 3’ end of the antisense strand. In some embodiments, the oligonucleotide comprises at least 2 phosphorothioate intemucleoside linkages at the 3’ end ofAttorney Docket No. 63578-717601 the antisense strand. In some embodiments, wherein the oligonucleotide is an RNAi agent, the at least one phosphorothioate intemucleoside linkage is at the 5’ end of the antisense strand. In some embodiments, the oligonucleotide comprises at least 2 phosphorothioate internucleoside linkages at the 5’ end of the sense strand. In some embodiments, the at least one phosphorothioate internucleoside linkage is at the 3’ end of the sense strand. In some embodiments, the oligonucleotide comprises at least 2 phosphorothioate internucleoside linkages at the 3’ end of the sense strand.

[0097] In some embodiments, any one of the oligonucleotides disclosed herein further comprises a terminal, chiral modification. In some embodiments, the terminal chiral modification is at the first internucleoside linkage at the 3’ end of the sense and / or antisense strand. In some embodiments, the terminal chiral modification is at the first and second internucleoside linkage at the 3’ end of the sense and / or antisense strand. In some embodiments, the chiral modification comprises a phosphorus atom of the intemucleoside linkage being in either Rp or Sp configuration. In some embodiments, the siRNA agent further comprises a terminal, chiral modification occurring at the first intemucleoside linkage at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first intemucleoside linkage at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first intemucleoside linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp configuration or Sp configuration. In some embodiments, the siRNA agent further comprises a terminal, chiral modification occurring at the first and second intemucleoside linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first intemucleoside linkage at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first intemucleoside linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration. In some embodiments, the siRNA agent further comprises a terminal, chiral modification occurring at the first, second, and third intemucleoside linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first intemucleoside linkage at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first intemucleoside linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration. In some embodiments, the siRNA agent further comprises a terminal, chiral modification occurring at the first and second intemucleoside linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal,Attorney Docket No. 63578-717601 chiral modification occurring at the third intemucleoside linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, a terminal, chiral modification occurring at the first internucleoside linkage at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first intemucleoside linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration. In some embodiments, the siRNA agent further comprises a terminal, chiral modification occurring at the first and second intemucleoside linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first and second intemucleoside linkages at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first intemucleoside linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

[0098] In some embodiments, any one of the oligonucleotides disclosed herein further comprises a phosphate or phosphate mimic at the 5’-end of the antisense strand. In some embodiments, a 5 ’-vinyl phosphonate (VP).RNAi Agent Modification Motifs

[0099] In some embodiments, any one of the antisense or sense strands disclosed herein are modified according to a modification motif or pattern.

[0100] In some embodiments, a strand of an RNAi agent comprises a modification pattern according to Nfsa(nNf)bnscn, wherein n is a 2'-O-methyl-nucleoside-3’ -phosphate; Nfs is a 2'-deoxy-2'-fluoro-nucleoside-3'-phosphorothioate; Nf is a 2'-deoxy-2'-fluoro-nucleoside-3'- phosphate; and ns is a 2'-O-methyl-nucleoside-3'-phosphorothioate; and wherein a is at least 1, b is at least 5-10, and c is at least 1. In some embodiments, a is at least 2, b is at least 8, and c is at least 2. In some embodiments, a is 2, b is 8, and c is 2. In some embodiments, the strand of the RNAi agent comprises the modification pattern NfsNfsnNfnNfnNfnNfnNfnNfnNfnNfnsnsn. In some embodiments, the strand is the antisense strand. In some embodiments, the antisense strand comprises the sequence of any one of SEQ ID NOs: 1540-2052, or any one of SEQ ID NOs: 1540-2052 or 2247-2335. In some embodiments, the strand is the sense strand. In some embodiments, the sense strand comprises the sequence of any one of SEQ ID NOs: 1027-1539, or any one of SEQ ID NOs: 1027-1539 or 2183-2246.

[0101] In some embodiments, a strand of an RNAi agent comprises a modification pattern according nsd(Nfn)eNf, wherein n is a 2'-O-methyl-nucleoside-3’ -phosphate; Nf is a 2'- deoxy-2'-fluoro-nucleoside-3'-phosphate; and ns is a 2'-O-methyl-nucleoside-3'- phosphorothioate; and wherein d is at least 1, and e is at least 5-10. In some embodiments, d is atAttorney Docket No. 63578-717601 least 2 and e is at least 8. In some embodiments, the RNAi agent comprises the modification pattern nsnsNfnNfnNfnNfnNfnNfnNfnNfnNf. In some embodiments, the strand is the sense strand. In some embodiments, the sense strand comprises the sequence of any one of SEQ ID NOs: 1027-1539, or any one of SEQ ID NOs: 1027-1539 or 2183-2246. In some embodiments, the strand is the antisense strand. In some embodiments, the antisense strand comprises the sequence of any one of SEQ ID NOs: 1540-2052, or any one of SEQ ID NOs: 1540-2052 or 2247-2335.

[0102] In some embodiments, the antisense strand comprises a modification pattern according to Nfsa(nNf)bnscn, wherein n is a 2'-O-methyl-nucleoside-3’ -phosphate; Nfs is a 2'- deoxy-2'-fluoro-nucleoside-3'-phosphorothioate; Nf is a 2'-deoxy-2'-fluoro-nucleoside-3'- phosphate; and ns is a 2'-O-methyl-nucleoside-3'-phosphorothioate; and wherein a is at least 1, b is at least 5-10, and c is at least 1, and the sense strand comprises a modification pattern according nsd(Nfn)eNf, wherein n is a 2'-O-methyl-nucleoside-3’ -phosphate; Nf is a 2'-deoxy-2'- fluoro-nucleoside-3 '-phosphate; and ns is a 2'-O-methyl-nucleoside-3'-phosphorothioate; and wherein d is at least 1, and e is at least 5-10.

[0103] In some embodiments, any one of the RNAi agents disclosed herein comprises a modification pattern wherein the antisense strand comprises the motif F(SF)nSnn, wherein n is from 2 to about 20, nn is 0 or 1, one of F and S is a 2 '-deoxy-2’ -fluoro modified nucleoside and the other of F and S is a 2'-O-methyl modified nucleoside. In some embodiments, each of the antisense and the sense strand is 17-23 nucleotides in length. In some embodiments, the antisense strand corresponds to the unmodified nucleobase sequence according to any one of SEQ ID NOs: 514-1026, or any one of SEQ ID NOs: 514-1026 or 2119-2182. In some embodiments, the sense strand corresponds to the unmodified nucleobase sequence according to any one of SEQ ID NOs: 1-513, or any one of SEQ ID NOs: 1-513 or 2055-2118.

[0104] In some embodiments, any one of the RNAi agents disclosed herein comprises a modification pattern wherein the antisense strand comprises the chemical modification pattern of nNfnnnNfnNfNfnnnnNfnNfnnnnnnn and the sense strand comprises the chemical modification nnnnnnNfnNfNfNfnnnnnnnnnn, wherein n is a 2’-O-methyl-nucleoside and Nf is a 2’-deoxy-2’- fluoro-nucleoside. In some embodiments, the antisense strand corresponds to the unmodified nucleobase sequence according to any one of SEQ ID NOs: 514-1026, or any one of SEQ ID NOs: 514-1026 or 2119-2182. In some embodiments, the sense strand corresponds to the unmodified nucleobase sequence of any one according to SEQ ID NOs: 1-513, or any one of SEQ ID NOs: 1-513 or 2055-2118.

[0105] In some embodiments, any one of the RNAi agents disclosed herein comprises a modification pattern wherein the antisense strand comprises a region having the formula Xl-Y-Attorney Docket No. 63578-717601X2, wherein Y is a region of from about 5 to about 12 linked nucleosides and each of XI and X2 is, independently, a plurality of linked nucleosides having the formula FSFS, where one of F and S is a 2 '-deoxy-2’ -fluoro modified nucleoside and the other of F and S is a 2'-O-methyl modified nucleoside; and each intemucleoside linkage of said first and said second oligomeric compound is, independently, a phosphodiester or a phosphorothioate internucleoside linkage. In some embodiments, each of the antisense and the sense strand is 20-23 nucleotides in length. In some embodiments, the antisense strand corresponds to the unmodified nucleobase sequence according to any one of SEQ ID NOs: 514-1026, or any one of SEQ ID NOs: 514-1026 or 2119-2182. In some embodiments, the sense strand corresponds to the unmodified nucleobase sequence according to any one of SEQ ID NOs: 1-513, or any one of SEQ ID NOs: 1-513 or 2055-2118.

[0106] In some embodiments, any one of the RNAi agents disclosed herein comprises a modification pattern wherein the antisense strand comprises a contiguous sequence of linked nucleosides that define an alternating motif of the formula: 5 '-Q(-L-Z-L-Q)n(-L-Z)nn-3', wherein: each L is an internucleoside linking group; either each Q is a 2 '-deoxy-2 ’-fluoro nucleoside and each Z is a 2'-O-methyl nucleoside; or each Q is a 2'-O-methyl nucleoside and each Z is a 2'-deoxy-2’-fluoro nucleoside; and n is from 8 to 14 and nn is 0 or 1. In some embodiments, the antisense strand corresponds to the unmodified nucleobase sequence according to any one of SEQ ID NOs: 514-1026, or any one of SEQ ID NOs: 514-1026 or 2119-2182.

[0107] In some embodiments, any one of the RNAi agents disclosed herein comprises a modification pattern wherein the antisense strand is represented by the formula:wherein, Bl', B2', B3', and B4' each independently represent a nucleotide containing a modification selected from the group consisting of 2'-O-alkyl, 2 '-substituted alkoxy, 2'- substituted alkyl, 2'-halo, ENA, and BNA / LNA; T1 ', T2', and T3' each independently represent a nucleotide comprising a chemical modification selected from the group consisting of DNA, RNA, LNA, 2’-deoxy-2’-fluoronucleotide, and 5'-(S)-methyl2’-deoxy-2’-fluoronucleotide; ql is independently 4 to 15 nucleotides in length; q3 or q7 is independently 1-6 nucleotide(s) in length; q2 or q6 is independently 1-3 nucleotide(s) in length; q4 is independently 0-3 nucleotide(s) in length; and q5 is independently 0-10 nucleotide(s) in length; and wherein: the antisense strand has 2’ -deoxy-2’ -fluoro modifications, and wherein the 2’ -deoxy-2’ -fluoro modifications on the antisense strand consist of four, and only four, 2 ’-deoxy-2 ’-fluoroAttorney Docket No. 63578-717601 modifications or six, and only six, 2 ’-deoxy-2’ -fluoro modifications. In some embodiments, the antisense strand is 19-25 nucleotides in length. In some embodiments, the antisense strand corresponds to the unmodified nucleobase sequence according to any one of SEQ ID NOs: 514- 1026, or any one of SEQ ID NOs: 514-1026 or 2119-2182. In some embodiments, the sense strand corresponds to the unmodified nucleobase sequence according to any one of SEQ ID NOs: 1-513, or any one of SEQ ID NOs: 1-513 or 2055-2118.

[0108] In some embodiments, any one of the RNAi agents disclosed herein comprises a modification pattern wherein the antisense strand and the sense strand is represented by the formula:wherein Bl, Bl', B2', B3', and B4' each independently represent a nucleotide containing a modification selected from the group consisting of 2'-O-alkyl, 2 '-substituted alkoxy, 2'- substituted alkyl, 2'-halo, ENA, and BNA / LNA; Cl is a thermally destabilizing nucleotide, selected from the group consisting of i) a nucleotide that forms a mismatch pair with the opposing nucleotide in the antisense strand, ii) a nucleotide having an abasic modification, and iii) a nucleotide having a sugar modification, and placed at a site opposite to the seed region (positions 2-8) of the antisense strand; T1 ', T2', and T3' each independently represent a nucleotide comprising a modification providing the nucleotide a steric bulk that is less than or equal to the steric bulk of a 2'-0Me modification, wherein the modification is at the 2'-position of a ribose sugar of the nucleotide or at a position of a non-ribose nucleotide similar to the 2'- position of a ribose sugar; each nl, and ql is independently 4 to 15 nucleotides in length; each q3, and q7 is independently 1-6 nucleotide(s) in length; each q2 and q6 is independently 1-3 nucleotide(s) in length; q5 is independently 0-10 nucleotide(s) in length; each n4, and q4 is independently 0-3 nucleotide(s) in length; n2 is 3 nucleotides in length, and T1 each are 2’- deoxy-2’ -fluoronucleotides; n3 is 7 nucleotides in length, and B2 each are 2'-0Me nucleotides; and n5 is 3 nucleotides in length, and B3 each are 2'-0Me nucleotides. In some embodiments, the antisense strand and sense strand are each 14 to 40 nucleotides. In some embodiments, the antisense strand corresponds to the unmodified nucleobase sequence according to any one of SEQ ID NOs: 514-1026, or any one of SEQ ID NOs: 514-1026 or 2119-2182. In someAttorney Docket No. 63578-717601 embodiments, the sense strand corresponds to the unmodified nucleobase sequence according to any one of SEQ ID NOs: 1-513, or any one of SEQ ID NOs: 1-513 or 2055-2118.

[0109] In some embodiments, any one of the RNAi agents disclosed herein comprises a modification pattern wherein the antisense strand and the sense strand is represented by the formula:(Is)wherein: Bl, B2, and B3 each independently represent a nucleotide containing a modification selected from the group consisting of 2'-O-alkyl, 2 '-substituted alkoxy, 2 '-substituted alkyl, 2'- halo, ENA, and BNA / LNA; Cl is a thermally destabilizing nucleotide, selected from the group consisting of i) a nucleotide that forms a mismatch pair with the opposing nucleotide in the antisense strand, ii) a nucleotide having an abasic modification, and iii) a nucleotide having a sugar modification, and placed at a site opposite to the seed region (positions 2-8) of the antisense strand T1 represents a nucleotide comprising a 2 ’-deoxy-2’ -fluoro modification; nl or n3 is independently 4 to 15 nucleotides in length; n5 is 1-6 nucleotide(s) in length; n2 is 3; n4 is 0-3 nucleotide(s) in length; and wherein the sense strand has 2’ -deoxy-2’ -fluoro modifications, and wherein the 2 ’-deoxy-2 ’-fluoro modifications on the sense strand consist of four, and only four, 2’ -deoxy-2’ -fluoro modifications, wherein the four 2 ’-deoxy-2 ’-fluoro modifications are at positions 7 and 9-11 from the 5 '-end of the sense strand. In some embodiments, wherein the antisense strand and sense strand are each 19-25 nucleotides. In some embodiments, the antisense strand corresponds to the unmodified nucleobase sequence according to any one of SEQ ID NOs: 514-1026, or any one of SEQ ID NOs: 514-1026 or 2119-2182. In some embodiments, the sense strand corresponds to the unmodified nucleobase sequence according to any one of SEQ ID NOs: 1-513, or any one of SEQ ID NOs: 1-513 or 2055-2118.

[0110] In some embodiments, any one of the RNAi agents disclosed herein comprises a modification pattern wherein the antisense strand is complementary to at least one portion of a mRNA of the target gene (e.g., GLIS2), wherein the oligonucleotide is represented by the formula:Attorney Docket No. 63578-717601 wherein: Bl', B2', B3', and B4' each independently represent a nucleotide containing a modification selected from the group consisting of 2'-0 -methyl and 2’ -deoxy-2’ -fluoro; each Bl, B2, and B3 is 2'-0Me; Cl is glycerol nucleic acid (GNA) placed at a site opposite to the seed region (positions 2-8) of the antisense strand; T1 ', T2', and T3' are each 2’ -deoxy-2’ - fluoronucleotides, wherein: T1 ' is at position 14 from the 5' end of the antisense strand, and q2 is 1; and T3' is at position 2 from the 5' end of the antisense strand, and q6 and q7 are 1; each nl, n3, and ql is independently 4 to 15 nucleotides in length; each n5 and q3 is independently 1-6 nucleotide(s) in length; q5 is independently 0-10 nucleotide(s) in length; each n4 and q4 is independently 0-3 nucleotide(s) in length; n2 is 3 nucleotides in length, and T1 each are 2’- deoxy-2’ -fluoro nucleotides, and wherein (a) the oligonucleotide is covalently conjugated to at least one ligand; and (b) one of the T1 nucleotides is at position 11 from the 5' end of the sense strand. In some embodiments, the sense strand has 19-22 nucleotides and the antisense strand has 19-25 nucleotides. In some embodiments, the antisense strand corresponds to the unmodified nucleobase sequence according to any one of SEQ ID NOs: 514-1026, or any one of SEQ ID NOs: 514-1026 or 2119-2182. In some embodiments, the sense strand corresponds to the unmodified nucleobase sequence according to any one of SEQ ID NOs: 1-513, or any one of SEQ ID NOs: 1-513 or 2055-2118.

[0111] In some embodiments, any one of the RNAi agents disclosed herein comprises a modification pattern wherein the oligonucleotide is represented by the formula:(i)wherein Bl', B2', B3', and B4' each independently represent a nucleotide containing a modification selected from the group consisting of 2'-O-methyl and 2 ’-deoxy-2 ’-fluoro; each Bl, B2, and B3 is 2'-0me nucleotides; Cl is glycerol nucleic acid (GNA) placed at a site opposite to the seed region (positions 2-8) of the antisense strand; T1 ', T2', and T3' are each 2’-deoxy-2’- fluoro nucleotides, wherein: T1 ' is at position 14 from the 5' end of the antisense strand, and q2 is 1; and T3' is at position 2 from the 5' end of the antisense strand, and q6 and q7 are 1; each nl, n3, and ql is independently 4 to 15 nucleotides in length; each n5 and q3 is independently 1-6 nucleotide(s) in length; q5 is independently 0-10 nucleotide(s) in length; each n4 and q4 is independently 0-3 nucleotide(s) in length; n2 is 3 nucleotides in length, and T1 each are 2’- deoxy-2’ -fluoro nucleotides, and wherein (a) the oligonucleotide is covalently conjugated to atAttorney Docket No. 63578-717601 least one ligand; and (b) one of the T1 nucleotides is at a position in the sense strand that is opposite to position 11 from the 5' end of the antisense strand; and (c) the oligonucleotide comprises at least one phosphorothioate intemucleoside linkage. In some embodiments, the sense strand has 19-22 nucleotides and the antisense strand has 19-25 nucleotides. In some embodiments, the antisense strand corresponds to the unmodified nucleobase sequence according to any one of SEQ ID NOs: 514-1026, or any one of SEQ ID NOs: 514-1026 or 2119-2182. In some embodiments, the sense strand corresponds to the unmodified nucleobase sequence according to any one of SEQ ID NOs: 1-513, or any one of SEQ ID NOs: 1-513 or 2055-2118.

[0112] In some embodiments, any one of the RNAi agents disclosed herein comprises a modification pattern wherein the antisense strand has sufficient complementarity to a target sequence to mediate RNA interference, wherein said sense strand comprises at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5' region of the antisense strand or a precursor thereof, wherein the antisense strand further comprises one or both of the following characteristics: (i) 2, 3, 4, 5 or 6 2 '-deoxy -2 '-fluoro modifications; and (ii) 1, 2, 3, 4 or 5 phosphorothioate internucleoside linkages; and said sense strand comprises one, two or three of the following characteristics: (iii) 2, 3, 4, or 5 2'-deoxy-2'- fluoro modifications; and (iv) 1, 2, 3, 4 or 5 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand and sense strand are each 14 to 40 nucleotides. In some embodiments, the antisense strand corresponds to the unmodified nucleobase sequence according to any one of SEQ ID NOs: 514-1026, or any one of SEQ ID NOs: 514-1026 or 2119-2182. In some embodiments, the sense strand corresponds to the unmodified nucleobase sequence according to any one of SEQ ID NOs: 1-513, or any one of SEQ ID NOs: 1-513 or 2055-2118.Pharmaceutical Compositions

[0113] The present disclosure also includes pharmaceutical compositions and formulations comprising the oligonucleotides (e.g., an RNAi agent or ASO) described herein. In one embodiment, provided herein are pharmaceutical compositions comprising an oligonucleotide (e.g., an RNAi agent or ASO), as described herein, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical compositions comprising the oligonucleotides described herein are useful for treating or preventing a condition or symptoms associated with GLIS2 expression. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery. The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of the target gene.Attorney Docket No. 63578-717601

[0114] A “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more oligonucleotides to a subject. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with the oligonucleotide and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, com starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

[0115] Formulations for topical administration of oligonucleotides can include sterile and non- sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the oligonucleotides in liquid or solid oil bases. The solutions can also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0116] In some embodiments, the oligonucleotides herein are conjugated to one or more non- nucleotide groups including, but not limited to, a targeting group / targeting moiety, linking group, delivery polymer, or a delivery vehicle. The non-nucleotide group can enhance targeting, delivery, or attachment of the oligonucleotide.

[0117] The non-nucleotide group can be covalently linked to the 3' and / or 5' end of either the antisense strand and / or the sense strand, when present. In some embodiments, when the oligonucleotide is an RNAi agent, the RNAi agent contains a non-nucleotide group linked to the 3' and / or 5' end of the sense strand. In some embodiments, a non-nucleotide group is linked to the 5' end of a GLIS2 RNAi agent sense strand. A non-nucleotide group may be linked directly or indirectly to the oligonucleotide via a linker / linking group. In some embodiments, a non- nucleotide group is linked to the oligonucleotide via a labile, cleavable, or reversible bond or linker.Attorney Docket No. 63578-717601

[0118] In some embodiments, the targeting moiety is a targeting ligand. In some embodiments, the targeting ligand is small molecule-based, saccharide-based, fatty acid-based, protein-based, or nucleic acid-based targeting ligand. In some embodiments, the targeting moiety is a protein-based targeting ligand. In some embodiments, the protein-based targeting ligand is an antibody, nanobody, affibody, or a peptibody.Methods of Treatment

[0119] The present disclosure also provides methods of using any one of the oligonucleotides disclosed herein to reduce or inhibit GLIS2 expression in a subject. The methods include contacting one or more cells in a subject with an oligonucleotide of the disclosure, thereby inhibiting expression of GLIS2 in the cell.

[0120] Reduction in GLIS2 expression can be assessed by any methods known in the art. For example, a reduction in the expression of GLIS2 may be determined by determining the mRNA expression level of GLIS2 using methods routine to one of ordinary skill in the art, e.g., northern blotting, qRT-PCR; by determining the protein level of GLIS2 using methods routine to one of ordinary skill in the art, such as western blotting, immunological techniques. In some embodiments, the reduction of GLIS2 expression is determined by measuring GLIS2 expression in a population of kidney cells derived from the subject.

[0121] The present disclosure further provides methods of treatment of a subject in need thereof. The methods of treatment include administering an oligonucleotide of the disclosure to a subject, e.g., a subject that would benefit from inhibition of GLIS2 expression, in a therapeutically effective amount of an oligonucleotide targeting an GLIS2 gene or a pharmaceutical composition comprising an oligonucleotide targeting an GLIS2 gene.

[0122] The in vivo methods of the disclosure may include administering to a subject a composition containing an oligonucleotide disclosed herein, wherein the oligonucleotide includes a nucleotide sequence that is complementary to at least a part of a sequence of nucleotides encoding GLIS2 of the subject to be treated.

[0123] Non-limiting examples of GLIS2 related disorders or disorders where inhibition of GLIS2 has utility for treatment with the oligonucleotides described herein include, but are not limited to, a polycystic kidney disease, such as an autosomal dominant polycystic kidney disease such as a polycystic kidney disease caused by or involving a mutation in the PKD I gene or the PKD2 gene.

[0124] The oligonucleotide can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, intravitreal,Attorney Docket No. 63578-717601 subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection. In certain embodiments, the compositions are administered by intrathecal injection. The administration of the oligonucleotide may be repeated over a period of time. The administration may be repeated on a regular basis. In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. A repeatdose regimen may include administration of a therapeutic amount of an oligonucleotide on a regular basis, such as monthly or extending to once a quarter, twice per year, once per year. In certain embodiments, the RNAi agent is administered about once per month to about once per quarter (z.e., about once every three months).

[0125] An oligonucleotide of the disclosure may be administered as a “free oligonucleotide.” A free oligonucleotide is administered in the absence of a pharmaceutical composition. The naked oligonucleotide may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the oligonucleotide can be adjusted such that it is suitable for administering to a subject. Alternatively, an RNAi agent of the disclosure may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

[0126] In some embodiments, the methods herein may further comprise administering to the subject an additional agent or therapy suitable for treatment or prevention of an GLIS2 related disorder. Non-limiting examples of such additional agents or therapies include any diuretic from any diuretic class (thiazides, loops, potassium-sparing, osmotic, carbonic anhydrase inhibitors, mineralocorticoid receptor antagonists), acetylcholinesterase inhibitors, angiotensin receptor blockers, neutral endopeptidase inhibitors, dual angiotensin receptor antagonists and neutral endopeptidase inhibitors, aldosterone antagonists, natriuretic peptides, calcium channel blockers, relaxin or relaxin mimetics, inotropic agents, peripheral vasodilators, or mineralocorticoid receptor antagonists.

[0127] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0128] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.”Attorney Docket No. 63578-717601

[0129] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0130] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.EXAMPLES

[0131] The following examples are provided to further illustrate some embodiments of the present disclosure, but are not intended to limit the scope of the disclosure; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.Example 1: GLIS2 siRNA Design and Evaluation

[0132] A set of siRNAs were designed to target GLIS2 (shown here as SEQ ID NOs: 2053-2054. A detailed list of the unmodified siRNA sense and antisense strand sequences is shown in Table 1 A below (with T representing uracil in the RNA sequences). A detailed list of modified siRNAs is shown in Table IB below. Table 2 provides abbreviations of modified nucleotides used in the nucleic acid sequences herein. Oligonucleotides are chemically synthesized using phosphoramidite approach and after quality control using UV- and massspectroscopy sense and antisense strands were annealed to perform screening

[0133] HeLa cells (ATCC, CCL-2) cells were maintained by biweekly passing in EMEM supplemented with 10% FBS, 20 mM L-glutamine, 10 mM HEPES pH 7.2, 1 mM sodium pyruvate, lx MEM non-essential amino acids. HeLa cells were purchased and cultured as described. Briefly, each vial of cells was thawed in 37 °C water bath and transferred to a 50 mL centrifuge tube filled with complete EMEM. The tube was then centrifuged at 200g for 5Attorney Docket No. 63578-717601 minutes, the media aspirated, and the cells resuspended to appropriate density and seeded for continuous culture.

[0134] One day before the transfection, HeLa cells were dissociated and seeded into 96 well plates at 10,000 cells / well in 100 pL of complete media. On the next day, the transfection mixes were prepared in the microcentrifuge tube by adding 10 ul of 100 nM siRNA to 15 ul of fresh OptiMEM (ThermoFisher, 31985062) in a v-bottom polypropylene 96 well plate (USA Scientific, 1833-9610). A master mix of Lipofectamine RNAiMax (ThermoFisher, 13778150) and OptiMEM media was created at a ratio of 0.3 pL RNAiMax and 25 pL of OptiMEM per reaction. 25 pL of RNAiMax master mix was added to each 25 pL of siRNA / OptiMEM, mixed by pipetting and incubated 5-10 min at RT. Media was aspirated from the HeLa cells and 50 ul of OptiMEM was added. After that, 50 ul of siRNA and RNAiMax of each siRNA / RNAiMax was added to respective wells of the HeLa cells, in triplicate. This created a final concentration of 10 nM siRNA per well. At 24 hours post transfection, cells were harvested and RNA isolated.

[0135] Multiple dose validation screens were performed just as the initial screens with small changes to preparation and final siRNA dosing. Individual siRNA at 5 pM were diluted appropriately into OptiMEM media to make 100 nM, 1 nM and 10 pM pre-stocks. Serial dilutions 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, 0.0005, 0.0001, 0.00005 nM were prepared in the separate microcentrifugal tubes using fresh OptiMEM in 25 ul. Just as with the single dose screens, 25 pL of RNAiMax master mix (0.3 pL RNAiMax to 25 pL OptiMEM) was added to each diluted siRNA, mixed by pipetting and incubated 5-10 min at RT. Media was aspirated from the HeLa cells and 50 ul of OptiMEM was added. After that, 50 ul of siRNA and RNAiMax of each siRNA / RNAiMax was added to respective wells of the HeLa cells, in triplicate. At 24 hours post transfection, cells were harvested and RNA isolated.

[0136] 24 hours post transfection, cells were harvested, and RNA isolated using theDirect-zol RNA kit (Zymo Research, R2062) according to the manufacturer protocol. Reverse transcription reaction was done using 150 ng of RNA and Maxima First Strand cDNA Synthesis Kit for RT-qPCR (ThermoFischer, K1642) according to the manufacturer protocol. cDNA was diluted 3 times and 5 ul was used for qPCR reaction. A single qPCR assay was performed for each sample using a Glis2 Taqman probe set (Hs00261493_ml-FAM) multiplexed with a common GAPDH-VIC probe (ThermoFisher, 4326322E) according to the manufacturer instructions for a combined primer / probe set. Thermocycling and data acquisition was performed with Applied Biosystems QuantStudio 6 Real-Time PCR System.

[0137] Quantification of Target mRNA and Knockdown Efficiency (as shown in Table IB): The mRNA levels of the target gene and the efficiency of siRNA-mediated knockdownAttorney Docket No. 63578-717601 were assessed using relative quantification. Cq values for both the target and reference (housekeeping) genes were obtained via quantitative PCR (qPCR) for each sample. The relative expression of the target gene was calculated using the 2A(-ACq) method, where ACq represents the difference between the Cq values of the target and reference genes.

[0138] Determination of ICso for siRNA: ICso values as shown in Table IB were determined by plotting the percentage of target gene depletion against the logarithm of siRNA concentrations. A sigmoidal dose-response (variable slope) curve was fitted to the data using the Curve Fitting function in GraphPad Prism (version 7.0). The ICso value was defined as the siRNA concentration resulting in 50% gene knockdown.

[0139] Table 1 A - Exemplary sequences for sense strand and antisense strand.Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601

[0140] Table IB - Exemplary sequences for sense strand and antisense strandAttorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601

[0141] Table 2: List of abbreviations of modified nucleotides used in the nucleic acid sequences herein.Attorney Docket No. 63578-717601

[0142] Table 3 - Exemplary sequences of mRNA transcriptAttorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601Attorney Docket No. 63578-717601

[0143] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the present disclosure may be employed in practicing the present disclosure. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Attorney Docket No. 63578-717601CLAIMSWhat is claimed is:

1. An oligonucleotide for inhibiting expression of GLIS family zinc finger 2 (GLIS2), wherein the oligonucleotide comprises an antisense strand comprising at least 14 contiguous nucleotides substantially complementary to a sequence of nucleotides encoding GLIS2, with no more than 4 mismatched nucleotides.

2. The oligonucleotide of claim 1, wherein the antisense strand is completely complementary to the sequence of nucleotides encoding GLIS2.

3. The oligonucleotide of claim 1 or 2, wherein the sequence of nucleotides encoding GLIS2 comprises a nucleotide sequence having at least 90% nucleotide sequence identity to any one of SEQ ID NOs: 2053-2054.

4. The oligonucleotide of any one of claims 1-3, wherein the antisense strand is substantially complementary to a sequence of nucleotides encoding GLIS2.

5. The oligonucleotide of any one of claims 1-4, wherein the antisense strand is substantially complementary to a sequence of nucleotides corresponding to an untranslated region of the GLIS2 transcript.

6. The oligonucleotide of claim 5, wherein the antisense strand is completely complementary to a sequence of nucleotides corresponding to an untranslated region of the GLIS2 transcript.

7. The oligonucleotide of any one of claims 1-6, wherein the oligonucleotide is an RNAi agent.

8. The oligonucleotide of claim 7, wherein the oligonucleotide is a double stranded small interfering RNA, a short hairpin RNA, or a Dicer- substrate siRNA (DsiRNA).

9. The oligonucleotide of claim 8, wherein the oligonucleotide is a double-stranded small interfering RNA (siRNA) further comprising a sense strand, wherein the sense strand and antisense strand comprise a double stranded region.Attorney Docket No. 63578-71760110. The oligonucleotide of any one of claims 1-9, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides differing by no more than 4 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 514-1026 or 2119-2182, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of any one of SEQ ID NOs: 514-1026 or 2119-2182.

11. The oligonucleotide of claim 10, wherein the antisense strand differs by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 514-1026 or 2119- 2182.

12. The oligonucleotide of claim 10 or 11, wherein the antisense strand differs by no more than 2 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 514-1026 or 2119-2182.

13. The oligonucleotide of any one of claims 10-12, wherein the antisense strand differs by no more than 1 nucleotide from the nucleotide sequence of any one of SEQ ID NOs: 514- 1026 or 2119-2182.

14. The oligonucleotide of any one of claims 10-13, wherein the antisense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 514-1026 or 2119-2182.

15. The oligonucleotide of any one of claims 9-14, wherein the sense strand comprises a nucleotide sequence differing by no more than 4 nucleotides from the nucleotide sequence of any one of SEQ ID Nos: 1-513 or 2055-2118, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of any one of SEQ ID NOs: 1-513 or 2055-2118.

16. The oligonucleotide of claim 15, wherein the sense strand differs by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 1-513 or 2055- 2118.

17. The oligonucleotide of claim 15 or 16, wherein the sense strand differs by no more than 2 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 1-513 or 2055-Attorney Docket No. 63578-71760118. The oligonucleotide of any one of claims 15-17, wherein the sense strand differs by no more than 1 nucleotide from the nucleotide sequence of any one of SEQ ID NOs: 1-513 or 2055-2118.

19. The oligonucleotide of any one of claims 15-18, wherein the sense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 1-513 or 2055-2118.

20. The oligonucleotide of any one of claims 9-19, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides differing by no more than 4 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 514-1026 or 2119-2182, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of any one of SEQ ID NOs: 514-1026 or 2119-2182, and the sense strand comprises a nucleotide sequence at least 15 contiguous nucleotides differing by no more than 4 nucleotides from the nucleotide sequence of any one of SEQ ID Nos: 1-513 or 2055-2118, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of any one of SEQ ID NOs: 1- 513 or 2055-2118.

21. The oligonucleotide of any one of claims 9-20, wherein each strand is no more than 30 nucleotides in length.

22. The oligonucleotide of any one of claims 9-21, wherein at least one strand comprises a 3’ overhang of at least 1 nucleotide.

23. The oligonucleotide of any one of claims 9-22, wherein at least one strand comprises a 3’ overhang of at least 2 nucleotides.

24. The oligonucleotide of any one of claims 9-23, wherein at least one strand comprises a 5’ overhang of at least 1 nucleotide.

25. The oligonucleotide of any one of claims 9-24, wherein at least one strand comprises a 5’ overhang of at least 2 nucleotides.

26. The oligonucleotide of any one of claims 9-25, wherein the double stranded region is 15- 30 nucleotide pairs in length.Attorney Docket No. 63578-71760127. The oligonucleotide of claim 9-26, wherein the double stranded region is 15-23 nucleotide pairs in length.

28. The oligonucleotide of claim 9-27, wherein the double stranded region is 17-25 nucleotide pairs in length.

29. The oligonucleotide of claim 9-28, wherein the double stranded region is 19-23 nucleotide pairs in length.

30. The oligonucleotide of claim 9-29, wherein the double stranded region is 19-21 nucleotide pairs in length.

31. The oligonucleotide of any one of claims 9-30, wherein each strand is 19-30 nucleotides in length.

32. The oligonucleotide of any one of claims 9-31, wherein each strand is 19-23 nucleotides in length.

33. The oligonucleotide of any one of claims 9-32, wherein each strand is 19-21 nucleotides in length.

34. The oligonucleotide of any one of claims 9-33, wherein the antisense is 21 nucleotides in length and the sense strand is 19 nucleotides in length.

35. The oligonucleotide of any one of claims 1-7, wherein the oligonucleotide is a singlestranded antisense oligonucleotide (ASO).

36. The oligonucleotide of claim 35, wherein the ASO comprises a nucleic acid sequence comprising at least 14 contiguous nucleotides that differ by no more than 1, 2, 3, or 4 nucleotides from any one of SEQ ID NOs: 514-1026, 1540-2052, 2119-2182, or 2247- 2335.

37. The oligonucleotide of claim 35 or 36, wherein the ASO comprises a nucleic acid sequence comprising at least 14 contiguous nucleotides that differ by no more than 1, 2, 3 or 4 nucleotides from any one of SEQ ID NOs: 514-1026, 1540-2052, 2119-2182, or 2247-2335.Attorney Docket No. 63578-71760138. The oligonucleotide of any one of claims 1-37, wherein the oligonucleotide comprises at least one or more modifications.

39. The oligonucleotide of claim 38, wherein the at least one or more modifications is selected from a ribose modification, a backbone modification, or a nucleobase modification.

40. The oligonucleotide of claim 39, wherein the ribose modification comprises a locked nucleic acid (LNA), a tricyclo-DNA (tcDNA), 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'- methoxyethyl (2'-M0E), 2'-deoxy-2'-arabino-fluoro, 2'-O-benzyl, 2'-O-(methyl-4- pyridine), 2' cyclic ethyl (cET), phosphorodiamidate morpholino (PMO), glycol nucleic acid (GNA), or unlocked nucleic acid (UNA).

41. The oligonucleotide of claim 40, wherein the ribose modification comprises a 2'-deoxy- 2'-fluoro, 2'-O-methyl, glycol nucleic acid (GNA), unlocked nucleic acid (UNA), or a threose nucleic acid (TNA).

42. The oligonucleotide of any one of claims 39-41, wherein the ribose modification is a 2'- deoxy-2'-fluoro or 2'-O-methyl modification.

43. The oligonucleotide of claim 39, wherein the backbone modification comprises phosphorothioate, phosphorodithioate, methylphosphonate, methyoxypropyl- phosphonate, 5'-(E)-vinylphosphonate, 5'-methyl phosphate, 5'-phosphorothioate, or peptide nucleic acid (PNA).

44. The oligonucleotide of claim 43, wherein the backbone modification comprises a phosphorothi oate .

45. The oligonucleotide of any one of claims 43-44, wherein the backbone modification comprises a phosphorothioate modification.

46. The oligonucleotide of claim 39, wherein the nucleobase modification comprises 5- substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, 5- methylcytosine (5-Me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, N6-alkyl derivatives, N2-alkyl, 2-thiouracil, 2-thiothymine, 2-Attorney Docket No. 63578-717601 thiocytosine, 5-halouracil, cytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8- sulfhydryl, 8-thioalkyl, 8-hydroxy, 5-halo, 5-trifluoromethyl, N7-methylguanine, N7- methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3- deazaguanine, 3 -deazaadenine, or any combination thereof.

47. The oligonucleotide of claim 46, wherein the oligonucleotide comprises at least one modified nucleotide selected from the group consisting of a deoxy nucleotide, a 3’- terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-deoxy- 2'-fluoro modified nucleotide, a 2'-deoxy -modified nucleotide, a 2’ -5 ’-linked ribonucleotide (3’-RNA), a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2’-amino-modified nucleotide, a 2’-O-allyl-modified nucleotide, 2’-C-alkyl- modified nucleotide, 2’-hydroxyl-modified nucleotide, a 2’-O-(methoxy ethyl) modified nucleotide, a 2’-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate morpholino, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a nucleotide comprising a 5'-methylphosphonate group, a nucleotide comprising a 5’ phosphate or 5’ phosphate mimic, a nucleotide comprising vinyl phosphonate, a glycol nucleic acid (GNA), a glycol nucleic acid S-Isomer (S-GNA), a nucleotide comprising 2- hydroxymethyl-tetrahydrofurane-5-phosphate, a nucleotide comprising 2’- deoxythymidine-3 ’phosphate, a nucleotide comprising 2 ’-deoxyguanosine-3’ -phosphate; a cytidine-2'-phosphate, a guanosine-2' -phosphate, a uridine-2'-phosphate, an adenosine- 2'-phosphate, a 2'-O-hexadecyl-adenosine-3'-phosphate, a 2'-O-hexadecyl-cytidine-3'- phosphate, a 2'-O-hexadecyl-guanosine-3'-phosphate, and a 2'-O-hexadecyl-uridine-3'- phosphate, a 3'-3' inverted nucleotide linkage, a 5’ -5’ inverted nucleotide linkage, TNA and combinations thereof.

48. The oligonucleotide of claim 38, wherein the nucleotide modification is a deoxy nucleotide, a 3’-terminal deoxythymine (dT) nucleotide, a 3'-3' inverted nucleotide linkage, a 5 ’-5’ inverted nucleotide linkage, or a 5'-(E)-vinylphosphonate-2’-O- methyluridine-3 ’ -phosphate.Attorney Docket No. 63578-71760149. The oligonucleotide of claim 48, wherein the nucleotide modification is 5'-(E)- vinylphosphonate-2’-O-methyluridine-3 ’-phosphate.

50. The oligonucleotide of claim 38, wherein at least one of the modifications is a thermally destabilizing nucleotide modification.

51. The oligonucleotide of claim 50, wherein the thermally destabilizing nucleotide modification is selected from the group consisting of an abasic modification; a mismatch with the opposing nucleotide in the duplex; and destabilizing sugar modification, a 2’- deoxy modification, an acyclic nucleotide, an unlocked nucleic acids (UNA), and a glycerol nucleic acid (GNA).

52. The oligonucleotide of claim 38, wherein the modification comprises a short sequence of 3 ’-terminal deoxythymine nucleotide (dT).

53. The oligonucleotide of any one of claims 38-47, wherein the modifications on the nucleotides are 2’-O-methyl and 2 ’deoxy-2’ -fluoro modifications.

54. The oligonucleotide of any one of claims 38-53, wherein the oligonucleotide comprises at least one phosphorothioate intemucleoside or phosphorodithioate intemucleoside linkage.

55. The oligonucleotide of claim 54, wherein the oligonucleotide comprises 6-8 phosphorothioate intemucleoside linkages.

56. The oligonucleotide of claims 54 or 55, wherein the oligonucleotide comprises at least 1 phosphorothioate intemucleoside linkage at a 5’ end of the sense strand.

57. The oligonucleotide of any one of claims 54-56, wherein the oligonucleotide comprises at least 2 phosphorothioate intemucleoside linkage at a 5’ end of the sense strand.

58. The oligonucleotide of any one of claims 54-57, wherein the oligonucleotide comprises at least 1 phosphorothioate intemucleoside linkage at a 3’ end of the sense strand.

59. The oligonucleotide of any one of claims 54-58, wherein the oligonucleotide comprises at least 1 phosphorothioate intemucleoside linkage at a 5’ end of the antisense strand.Attorney Docket No. 63578-71760160. The oligonucleotide of any one of claims 54-59, wherein the oligonucleotide comprises at least 2 phosphorothioate internucleoside linkage at a 5’ end of the antisense strand.

61. The oligonucleotide of any one of claims 54-60, wherein the oligonucleotide comprises at least 1 phosphorothioate internucleoside linkage at a 3’ end of the sense antisense.

62. The oligonucleotide of any one of claims 38-61, wherein no more than five of the nucleotides of the antisense strand are unmodified nucleotides.

63. The oligonucleotide of any one of claims 38-62, wherein all the nucleotides of the antisense strand are modified oligonucleotides.

64. The oligonucleotide of any one of claims 38-63, wherein no more than five of the sense strand nucleotides are unmodified nucleotides.

65. The oligonucleotide of any one of claims 38-64, wherein all the nucleotides of the sense strand are modified nucleotides.

66. The oligonucleotide of any one of claims 1-65, wherein the antisense strand comprises a chemical modification pattern according to (Nfs)a(nNf)b(ns)cn, wherein: n is a 2'-O-methyl-nucleoside-3’ -phosphate;Nfs is a 2'-deoxy-2'-fluoro-nucleoside-3'-phosphorothioate;Nf is a 2'-deoxy-2'-fluoro-nucleoside-3'-phosphate; and ns is a 2'-O-methyl-nucleoside-3'-phosphorothioate, and wherein a is at least 1, b is at least 5-10, and c is at least 1.

67. The oligonucleotide of claim 66, wherein the antisense strand comprises the chemical modification pattern NfsNfsnNfnNfnNfnNfnNfnNfnNfnNfnsnsn.

68. The oligonucleotide of any one of claims 9-34, wherein the sense strand comprises a chemical modification pattern according to (ns)d(Nfn)eNf, wherein: n is a 2’-O-methyl-nucleoside-3 ’-phosphate;Attorney Docket No. 63578-717601Nf is a 2’-deoxy-2'-fluoro-nucleoside-3 ’-phosphate; and ns is a 2’-O-methyl-nucleoside-3’-phosphorothioate, and wherein d is at least 1 and e is at least 5-10.

69. The oligonucleotide of claim 68, wherein the sense strand comprises the chemical modification pattern nsnsNfnNfnNfnNfnNfnNfnNfnNfnNf.

70. The oligonucleotide of any one of claims 9-34, wherein each of the antisense and the sense strand is 17-23 nucleotides in length, wherein the antisense strand comprises the motif F(SF)nSnn, wherein n is from 2 to about 20, nn is 0 or 1, one of F and S is a 2'- deoxy-2’ -fluoro modified nucleoside and the other of F and S is a 2'-O-methyl modified nucleoside.

71. The oligonucleotide of any one of claims 9-34, wherein the antisense strand comprises the chemical modification pattern of nNfnnnNfnNfNfnnnnNfnNfnnnnnnn and the sense strand comprises the chemical modification nnnnnnNfnNfNfNfnnnnnnnnnn, wherein n is a 2’-O-methyl-nucleoside and Nf is a 2’-deoxy-2’-fluoro-nucleoside.

72. The oligonucleotide of any one of claims 9-34, wherein each of the antisense and the sense strand is 20-23 nucleotides in length, wherein the antisense strand comprises a region having the formula X1-Y-X2, wherein Y is a region of from about 5 to about 12 linked nucleosides and each of XI and X2 is, independently, a plurality of linked nucleosides having the formula FSFS, where one of F and S is a 2 '-deoxy-2’ -fluoro modified nucleoside and the other of F and S is a 2'-O-methyl modified nucleoside; and each intemucleoside linkage of said first and said second oligomeric compound is, independently, a phosphodiester or a phosphorothioate internucleoside linkage.

73. The oligonucleotide of any one of claims 9-34, wherein each of the antisense and the sense strand is 17-23 nucleotides in length, wherein the antisense strand comprises a contiguous sequence of linked nucleosides that define an alternating motif of the formula: 5 '-Q(-L-Z-L-Q)n(-L-Z)nn-3' wherein: each L is an internucleoside linking group; either each Q is a 2'-deoxy-2’-fluoro-nucleoside and each Z is a 2'-O-methyl nucleoside; or each Q is a 2'-O-methyl nucleoside and each Z is a 2 '-deoxy-2 ’-fluoro nucleoside; and n is from 8 to 14 and nn is 0 or 1.Attorney Docket No. 63578-71760174. The oligonucleotide of any one of claims 9-34, wherein the antisense strand is 19-25 nucleotides in length and is represented by the formula: fla)wherein:Bl', B2', B3', and B4' each independently represent a nucleotide containing a modification selected from the group consisting of 2'-O-alkyl, 2 '-substituted alkoxy, 2 '-substituted alkyl, 2'-halo, ENA, and BNA / LNA;T1 ', T2', and T3' each independently represent a nucleotide comprising a chemical modification selected from the group consisting of DNA, RNA, LNA, 2'-deoxy- 2’-fluoro, and 5 '-methyl-2'-deoxy-2’-fluoro nucleotides; ql is independently 4 to 15 nucleotides in length; q3 or q7 is independently 1-6 nucleotide(s) in length; q2 or q6 is independently 1-3 nucleotide(s) in length; q4 is independently 0-3 nucleotide(s) in length; and q5 is independently 0-10 nucleotide(s) in length; and wherein: the antisense strand has 2'-deoxy-2’-fluoro modifications, and wherein the 2'-deoxy-2’- fluoro modifications on the antisense strand consist of four, and only four, 2'-deoxy-2’-fluoro modifications or six, and only six, 2'-deoxy-2’-fluoro modifications.

75. The oligonucleotide of any one of claims 9-34, wherein the antisense strand and sense strand are each 14 to 40 nucleotides, and is represented by the formula:Attorney Docket No. 63578-717601wherein:Bl, Bl', B2', B3', and B4' each independently represent a nucleotide containing a modification selected from the group consisting of 2'-O-alkyl, 2 '-substituted alkoxy, 2'- substituted alkyl, 2'-halo, ENA, and BNA / LNA;Cl is a thermally destabilizing nucleotide, selected from the group consisting of i) a nucleotide that forms a mismatch pair with the opposing nucleotide in the antisense strand, ii) a nucleotide having an abasic modification, and iii) a nucleotide having a sugar modification, and placed at a site opposite to the seed region (positions 2-8) of the antisense strand;T1 ', T2', and T3' each independently represent a nucleotide comprising a modification providing the nucleotide a steric bulk that is less than or equal to the steric bulk of a 2'-0Me modification, wherein the modification is at the 2'-position of a ribose sugar of the nucleotide or at a position of a non-ribose nucleotide similar to the 2'- position of a ribose sugar; each nl, and ql is independently 4 to 15 nucleotides in length; each q3, and q7 is independently 1-6 nucleotide(s) in length; each q2 and q6 is independently 1-3 nucleotide(s) in length; q5 is independently 0-10 nucleotide(s) in length; each n4, and q4 is independently 0-3 nucleotide(s) in length; n2 is 3 nucleotides in length, and T1 each are 2 '-deoxy -2’ -fluoro nucleotides; n3 is 7 nucleotides in length, and B2 each are 2'-0Me nucleotides; and n5 is 3 nucleotides in length, and B3 each are 2'-0Me nucleotides.

76. The oligonucleotide of any one of claims 9-34, wherein the antisense strand and sense strand are each 19-25 nucleotides in length, wherein the sense strand is represented by the formula:Attorney Docket No. 63578-717601(Is)wherein:Bl, B2, and B3 each independently represent a nucleotide containing a modification selected from the group consisting of 2'-O-alkyl, 2 '-substituted alkoxy, 2 '-substituted alkyl, 2'- halo, ENA, and BNA / LNA;Cl is a thermally destabilizing nucleotide, selected from the group consisting of i) a nucleotide that forms a mismatch pair with the opposing nucleotide in the antisense strand, ii) a nucleotide having an abasic modification, and iii) a nucleotide having a sugar modification, and placed at a site opposite to the seed region (positions 2-8) of the antisense strand;T1 represents a nucleotide comprising a 2'-deoxy-2’ -fluoro modification; nl or n3 is independently 4 to 15 nucleotides in length; n5 is 1-6 nucleotide(s) in length; n2 is 3; n4 is 0-3 nucleotide(s) in length; and wherein the sense strand has 2'-deoxy-2’-fluoro modifications, and wherein the 2'-deoxy-2’- fluoro modifications on the sense strand consist of four, and only four, 2'-deoxy-2’-fluoro modifications, wherein the four 2'-deoxy-2’-fluoro modifications are at positions 7 and 9-11 from the 5 '-end of the sense strand.

77. The oligonucleotide of any one of claims 9-34, wherein the antisense strand is complementary to at least one portion of a mRNA of the target gene, wherein: the sense strand has 19-22 nucleotides, the antisense strand has 19-25 nucleotides; and the oligonucleotide is represented by the formula:(i)Attorney Docket No. 63578-717601 wherein:Bl', B2', B3', and B4' each independently represent a nucleotide containing a modification selected from the group consisting of 2'-O-methyl and 2 '-deoxy-2 ’ -fluoro; each Bl, B2, and B3 is 2'-O-methyl nucleotide;Cl is glycerol nucleic acid (GNA) placed at a site opposite to the seed region (positions 2-8) of the antisense strand;T1 ', T2', and T3' are each 2'-F, wherein:T1 ' is at position 14 from the 5' end of the antisense strand, and q2 is 1; andT3' is at position 2 from the 5' end of the antisense strand, and q6 and q7 are 1 ; each nl, n3, and ql is independently 4 to 15 nucleotides in length; each n5 and q3 is independently 1-6 nucleotide(s) in length; q5 is independently 0-10 nucleotide(s) in length; each n4 and q4 is independently 0-3 nucleotide(s) in length; n2 is 3 nucleotides in length, and T1 each are 2 '-deoxy-2 ’-fluoro nucleotides, and wherein(a) the oligonucleotide is covalently conjugated to at least one ligand; and(b) one of the T1 nucleotides is at position 11 from the 5' end of the sense strand.

78. The oligonucleotide of any one of claims 9-34, wherein the sense strand has 19-22 nucleotides, the antisense strand has 19-25 nucleotides; and the oligonucleotide is represented by the formula:(i)wherein:Bl', B2', B3', and B4' each independently represent a nucleotide containing a modification selected from the group consisting of 2'-O-methyl and 2 '-deoxy-2 ’-fluoro; each Bl, B2, and B3 is 2'-0Me;Cl is glycerol nucleic acid (GNA) placed at a site opposite to the seed region (positions 2-8) of the antisense strand;Attorney Docket No. 63578-717601T1 ', T2', and T3' are each 2 '-deoxy-2 ’-fluoro, wherein:T1 ' is at position 14 from the 5' end of the antisense strand, and q2 is 1; andT3' is at position 2 from the 5' end of the antisense strand, and q6 and q7 are 1; each nl, n3, and ql is independently 4 to 15 nucleotides in length; each n5 and q3 is independently 1-6 nucleotide(s) in length; q5 is independently 0-10 nucleotide(s) in length; each n4 and q4 is independently 0-3 nucleotide(s) in length; n2 is 3 nucleotides in length, and T1 each are 2 '-deoxy-2 ’-fluoro, and wherein(a) the oligonucleotide is covalently conjugated to at least one ligand; and(b) one of the T1 nucleotides is at a position in the sense strand that is opposite to position 11 from the 5' end of the antisense strand; and(c) the oligonucleotide comprises at least one phosphorothioate internucleoside linkage.

79. The oligonucleotide of any one of claims 9-34, wherein the antisense strand and sense strand are each 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to a target sequence to mediate RNA interference, wherein said sense strand comprises at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5' region of the antisense strand or a precursor thereof, wherein the antisense strand further comprises one or both of the following characteristics:(i) 2, 3, 4, 5 or 6 2 '-deoxy-2 ’-fluoro modifications; and(ii) 1, 2, 3, 4 or 5 phosphorothioate intemucleoside linkages; and said sense strand comprises one, two or three of the following characteristics:(iii) 2, 3, 4, or 5 2 '-deoxy-2 ’-fluoro modifications; and(iv) 1, 2, 3, 4 or 5 phosphorothioate intemucleoside linkages.

80. The oligonucleotide of any one of claims 9-79, wherein the antisense strand comprises a nucleotide sequence according to any one of SEQ ID NOs: 514-1026 or 2119-2182.

81. The oligonucleotide of claim 80, wherein the sense strand comprises a nucleotide sequence according to any one of SEQ ID NOs: 1-513 or 2055-2118.Attorney Docket No. 63578-71760182. The oligonucleotide of any one of claims 1-81, wherein administration of the oligonucleotide results in at least a 55% reduction in expression levels of an GLIS2 mRNA in kidney cells.

83. The oligonucleotide of any one of claims 1-82, wherein administration of the oligonucleotide results in at least a 75% reduction in expression levels of an GLIS2 mRNA in kidney cells.

84. The oligonucleotide of any one of claims 1-83, wherein administration of the oligonucleotide results in at least an 80% reduction in expression levels of an GLIS2 mRNA in kidney cells.

85. The oligonucleotide of any one of claims 1-84, wherein administration of the oligonucleotide results in at least an 85% reduction in expression levels of an GLIS2 mRNA in kidney cells.

86. The oligonucleotide of any one of claims 9-85, further comprising a terminal, chiral modification occurring at the first intemucleoside linkage at the 3 ’-end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first intemucleoside linkage at the 5 ’-end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first intemucleoside linkage at the 5 ’-end of the sense strand, having the linkage phosphorus atom in either Rp configuration or Sp configuration.

87. The oligonucleotide of any one of claims 9-85, further comprising a terminal, chiral modification occurring at the first and second intemucleoside linkages at the 3 ’-end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first intemucleoside linkage at the 5’-end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first intemucleoside linkage at the 5’-end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

88. The oligonucleotide of any one of claims 9-85, further comprising a terminal, chiral modification occurring at the first, second and third intemucleoside linkages at the 3’ endAttorney Docket No. 63578-717601 of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first intemucleoside linkage at the 5 ’-end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first intemucleoside linkage at the 5 ’-end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

89. The oligonucleotide of any one of claims 9-85, further comprising a terminal, chiral modification occurring at the first, and second intemucleoside linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the third intemucleoside linkages at the 3 ’-end of the antisense strand, having the linkage phosphorus atom in Rp configuration, a terminal, chiral modification occurring at the first intemucleoside linkage at the 5’-end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first intemucleoside linkage at the 5’-end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

90. The oligonucleotide of any one of claims 9-85, further comprising a terminal, chiral modification occurring at the first, and second intemucleoside linkages at the 3 ’-end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first, and second intemucleoside linkages at the 5’- end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first intemucleoside linkage at the 5 ’-end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

91. The oligonucleotide of any one of claims 9-85, further comprising a phosphate or phosphate mimic at the 5 ’-end of the antisense strand.

92. The oligonucleotide of claim 91, wherein the phosphate mimic is a 5 ’-vinyl phosphonate (VP).

93. A pharmaceutical composition comprising the oligonucleotide of any one of claims 1-92.

94. The pharmaceutical composition of claim 93, further comprising a targeting moiety.

95. The pharmaceutical composition of claim 94, wherein the targeting moiety is a targeting ligand.Attorney Docket No. 63578-71760196. The pharmaceutical composition of claim 95, wherein the targeting ligand is a small molecule-based, sugar-based, fatty acid-based, protein-based, or nucleic acid-based targeting ligand.

97. The pharmaceutical composition of claim 96, wherein the protein-based targeting ligand is an antibody, nanobody, affibody, peptibody, or a peptide.

98. A method for inhibiting GLIS2 expression in a subject, the method comprising administering an effective amount of the oligonucleotide of any one of claims 1-92 or the pharmaceutical composition of any one of claims 93-97 to the subject.

99. The method of claim 98, wherein the subject is a human.

100. The method of claim 98 or 99, wherein the reduction of GLIS2 mRNA or protein expression levels is measured in a population of kidney cells derived from the subject.

101. The method of any one of claims 98-100, wherein the reduction of GLIS2 mRNA or protein expression levels is measured in tissues derived from the subject.

102. The method of any one of claims 98-101, further comprising administering to the subject an additional agent or a therapy suitable for treatment or prevention of an GLIS2 related disorder.

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