WO2022026719A2 - Nrl antisense oligonucleotides, compositions containing the same, and methods of their use - Google Patents

Abstract

Disclosed are oligonucleotides having a nucleobase sequence with at least 6 contiguous nucleobases complementary to an equal-length portion within an NRL target nucleic acid. Also disclosed are pharmaceutical compositions containing the oligonucleotides and methods of their use.

Description

NRL ANTISENSE OLIGONUCLEOTIDES,

COMPOSITIONS CONTAINING THE SAME, AND METHODS OF THEIR USE

FIELD OF THE INVENTION

The invention provides oligonucleotides, compositions containing the same, and methods of their use.

BACKGROUND

Retinitis pigmentosa is a group of inherited, progressive diseases causing retinal degeneration. Patients having retinitis pigmentosa experience a gradual decline in their vision because photoreceptor cells in the retina degenerate.

In most forms of retinitis pigmentosa, rod cells are affected first. Because rods are concentrated in outer portions of the retina and are triggered by dim light, their degeneration affects peripheral and night vision. When the disease progresses and cones become affected, visual acuity, color perception, and central vision are diminished. Night blindness is one of the earliest and most frequent symptoms of retinitis pigmentosa. On the other hand, patients with cone degeneration first experience decreased central vision and reduced ability to discriminate colors and perceive details.

Retinitis pigmentosa is typically diagnosed in adolescents and young adults. The rate of progression and degree of visual loss varies from person to person. Most people with retinitis pigmentosa are legally blind by age 40 with a central visual field of less than 20 degrees in diameter.

There is currently no cure for retinitis pigmentosa. Applicability of various supplements, such as vitamin A, docosahexaenoic acid, and lutein, to slow the progression of retinitis pigmentosa remain largely unresolved. Currently, the main marketed treatment for retinitis pigmentosa is an electronic retinal implant. This treatment approach, however, requires intraocular, surgical implantation and is prosthetic by design. Therefore, it does not prevent the loss of rod and cone cells underlying the symptoms of retinitis pigmentosa.

There is a need for new therapeutic approaches to the treatment of retinitis pigmentosa.

SUMMARY OF THE INVENTION

In general, the invention provides oligonucleotides including a nucleobase sequence including at least 6 contiguous nucleobases complementary to an equal-length portion within a NRL target nucleic acid. The invention also provides compositions containing oligonucleotides of the invention and methods of using the same.

In one aspect, the invention provides a single-stranded oligonucleotide including a total of 12 to 50 interlinked nucleotides and having a nucleobase sequence including at least 6 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof (e.g., SEQ ID NOs: 18-29).

In another aspect, the invention provides a single-stranded oligonucleotide comprising a total of 12 to 50 interlinked nucleotides and having a nucleobase sequence comprising at least 6 contiguous nucleobases complementary to the target sequence of any one of SEQ ID NOs: 6-17. In some embodiments, the oligonucleotide includes at least one modified nucleobase. In some embodiments, at least one modified nucleobase is 5-methylcytosine. In some embodiments, at least one modified nucleobase is 7-deazaguanine. In some embodiments, at least one modified nucleobase is 6- thioguanine.

In some embodiments, the oligonucleotide includes at least one modified internucleoside linkage. In some embodiments, the modified internucleoside linkage is a phosphorothioate linkage. In some embodiments, the phosphorothioate linkage is a stereochemically enriched phosphorothioate linkage. In some embodiments, at least 50% of internucleoside linkages in the oligonucleotide are each independently the modified internucleoside linkage. In some embodiments, at least 70% of internucleoside linkages in the oligonucleotide are each independently the modified internucleoside linkage. In some embodiments, 100% of the internucleoside linkages in the oligonucleotide are the modified internucleoside linkage.

In some embodiments, the oligonucleotide includes at least one modified sugar nucleoside. In some embodiments, at least one modified sugar nucleoside is a bridged nucleic acid. In some embodiments, the bridged nucleic acid is a locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), or cEt nucleic acid. In some embodiments, the oligonucleotide is a gapmer. In some embodiments, at least one modified sugar nucleoside is a 2’-modified sugar nucleoside. In some embodiments, at least one 2’-modified sugar nucleoside includes a 2’-modification selected from the group consisting of 2’-fluoro, 2’-methoxy, and 2’-methoxyethoxy. In some embodiments, the oligonucleotide includes deoxyribonucleotides. In some embodiments, the oligonucleotide includes ribonucleotides.

In some embodiments, the oligonucleotide is a morpholino oligomer.

In some embodiments, the oligonucleotide includes a hydrophobic moiety covalently attached at a 5’-terminus, 3’-terminus, or internucleoside linkage of the oligonucleotide.

In some embodiments, the oligonucleotide includes a total of at least 7 interlinked nucleotides. In some embodiments, the single-stranded oligonucleotide includes at least 7 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof (e.g., SEQ ID NOs: 18-29).

In some embodiments, the oligonucleotide includes a total of at least 8 interlinked nucleotides. In some embodiments, the single-stranded oligonucleotide includes at least 8 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof (e.g., SEQ ID NOs: 18-29).

In some embodiments, the oligonucleotide includes a total of at least 9 interlinked nucleotides. In some embodiments, the single-stranded oligonucleotide includes at least 9 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof (e.g., SEQ ID NOs: 18-29).

In some embodiments, the oligonucleotide includes a total of at least 10 interlinked nucleotides.

In some embodiments, the single-stranded oligonucleotide includes at least 10 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof. In some embodiments, the oligonucleotide includes a total of 10 interlinked nucleotides or fewer. In some embodiments, the oligonucleotide includes a total of at least 11 interlinked nucleotides.

In some embodiments, the single-stranded oligonucleotide includes at least 11 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof (e.g., SEQ ID NOs: 18-29).

In some embodiments, the oligonucleotide includes a total of at least 12 interlinked nucleotides.

In some embodiments, the single-stranded oligonucleotide includes at least 12 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof (e.g., SEQ ID NOs: 18-29).

In some embodiments, the oligonucleotide includes a total of at least 13 interlinked nucleotides.

In some embodiments, the single-stranded oligonucleotide includes at least 13 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof (e.g., SEQ ID NOs: 18-29).

In some embodiments, the oligonucleotide includes a total of at least 14 interlinked nucleotides.

In some embodiments, the single-stranded oligonucleotide includes at least 14 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof (e.g., SEQ ID NOs: 18-29).

In some embodiments, the oligonucleotide includes a total of at least 15 interlinked nucleotides.

In some embodiments, the single-stranded oligonucleotide includes at least 15 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof (e.g., SEQ ID NOs: 18-29).

In some embodiments, the oligonucleotide includes a total of at least 16 interlinked nucleotides.

In some embodiments, the single-stranded oligonucleotide includes at least 16 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof (e.g., SEQ ID NOs: 18-29).

In some embodiments, the oligonucleotide includes a total of 25 interlinked nucleotides or fewer. In some embodiments, the oligonucleotide includes a total of 20 interlinked nucleotides or fewer. In some embodiments, the oligonucleotide includes a total of 19 interlinked nucleotides or fewer. In some embodiments, the oligonucleotide includes a total of 18 interlinked nucleotides or fewer. In some embodiments, the oligonucleotide includes a total of 17 interlinked nucleotides or fewer. In some embodiments, the oligonucleotide includes a total of 16 interlinked nucleotides or fewer. In some embodiments, the oligonucleotide includes a total of 15 interlinked nucleotides or fewer. In some embodiments, the oligonucleotide includes a total of 14 interlinked nucleotides or fewer. In some embodiments, the oligonucleotide includes a total of 13 interlinked nucleotides or fewer. In some embodiments, the oligonucleotide includes a total of 12 interlinked nucleotides or fewer. In some embodiments, the oligonucleotide includes a total of 11 interlinked nucleotides or fewer.

In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 6. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 7. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 8. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 9. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 10. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 11. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 12. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 13. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 14. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 15. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 16. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 17. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 18. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 19. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 20. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 21 . In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 22. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 23. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 24. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 25. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 26. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 27. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 28. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 29.

In some embodiments, the sequence of the oligonucleotide comprises or consists of the nucleotide sequence referenced in the claim.

In another aspect, the invention provides an oligonucleotide having a nucleotide sequence that comprises the sequence of any one of SEQ ID NOs: 6-29 or a chemically modified version thereof (e.g., SEQ ID NOs: 18-29).

In some embodiments, the oligonucleotide has a nucleotide sequence that consists of the sequence of any one of SEQ ID NOs: 6-29 or a chemically modified version thereof (e.g., SEQ ID NOs: 18-29).

In some embodiments, the oligonucleotide comprises a feature or modification as set forth above and elsewhere herein.

In some embodiments, the “chemically modified version thereof comprises a 5-methyl-dC nucleotide in place of C.

In some embodiments, a “chemically modified version thereof of any one of SEQ ID NOs: 6-17 is SEQ ID NO: 18-29, respectively.

In another aspect, the invention provides a pharmaceutically acceptable salt of an oligonucleotide as set forth above and elsewhere herein.

In another aspect, the invention provides a double-stranded oligonucleotide including an oligonucleotide of the invention hybridized to a complementary oligonucleotide. In some embodiments, the complementary oligonucleotide has the same length as the oligonucleotide of the invention. In further embodiments, the complementary oligonucleotide has a length that is at least, e.g., ±1 , ±2, ±3, ±4, or ±5 nucleotides relative to the number of nucleotides in the oligonucleotide of the invention.

In another aspect, the invention provides a pharmaceutical composition including the oligonucleotide of the invention and a pharmaceutically acceptable excipient.

In a yet another aspect, the invention provides methods of use of the oligonucleotides of the invention.

In some embodiments, the method is a method of inhibiting or reducing the production of an NRL protein in a cell including (e.g., expressing) an NRL gene by contacting the cell with the oligonucleotide of the invention. In certain embodiments, the cell is in a subject. In particular embodiments, the cell is in the subject’s eye.

In further embodiments, the method is a method of treating a subject in need thereof by administering to the subject a therapeutically effective amount of the oligonucleotide of the invention or the pharmaceutical composition of the invention.

In yet further embodiments, the oligonucleotide or pharmaceutical composition is administered intraocularly or topically to the eye of the subject. In still further embodiments, the subject is in need of a treatment for an ocular disease, disorder, or condition associated with a dysfunction of ABCA4, AIPL1 , BBS1 , BEST1 , CEP290, CDH3, CHM, CNGA3, CNGB3, CRB1 , GUCY2D, MERTK, MRFP, MY07A,

ND4, NR2E3, PDE6, PRPH2, RD3, RHO, RLBP1 , RP1 , RPE65, RPGR, RPGRIP1 , RS1 , or SPATA7 gene. In some embodiments, the subject is in need of a treatment for retinitis pigmentosa, Stargardt disease, cone-rod dystrophy, Leber congenital amaurosis, Bardet Biedl syndrome, macular dystrophy, dry macular degeneration, geographic atrophy, atrophic age-related macular degeneration (AMD), advanced dry AMD, retinal dystrophy, choroideremia, Usher syndrome type 1 , retinoschisis, Leber hereditary optic neuropathy, and achromatopsia. In preferred embodiments, the subject is in need of a treatment for retinitis pigmentosa. In certain embodiments, retinitis pigmentosa is Rho P23H-associated retinitis pigmentosa, PDE6-associated retinitis pigmentosa, MERTK-associated retinitis pigmentosa, BBS1- associated retinitis pigmentosa, Rho-associated retinitis pigmentosa, MRFP-associated retinitis pigmentosa, RLBP1 -associated retinitis pigmentosa, RP1 -associated retinitis pigmentosa, RPGR-X- linked retinitis pigmentosa, NR2E3-associated retinitis pigmentosa, or SPATA7-associated retinitis pigmentosa.

The invention also provides the oligonucleotides described herein, and corresponding salts and/or compositions, for use in treating the diseases and conditions described herein, as well as for inhibiting or reducing production of an NRL protein in a cell as described herein.

Definitions

The term “acyl,” as used herein, represents a chemical substituent of formula -C(0)-R, where R is alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, heterocyclyl alkyl, heteroaryl, or heteroaryl alkyl. An optionally substituted acyl is an acyl that is optionally substituted as described herein for each group R.

The term “acyloxy,” as used herein, represents a chemical substituent of formula -OR, where R is acyl. An optionally substituted acyloxy is an acyloxy that is optionally substituted as described herein for acyl.

The term “aliphatic,” as used herein, refers to an acyclic, branched or acyclic, linear hydrocarbon chain, or a monocyclic, bicyclic, tricyclic, or tetracyclic hydrocarbon. Unless specified otherwise, an aliphatic group includes a total of 1 to 60 carbon atoms. An optionally substituted aliphatic is an optionally substituted acyclic aliphatic or an optionally substituted cyclic aliphatic. An optionally substituted acyclic aliphatic is optionally substituted as described herein for alkyl. An optionally substituted cyclic aliphatic is an optionally substituted aromatic aliphatic or an optionally substituted non-aromatic aliphatic. An optionally substituted aromatic aliphatic is optionally substituted as described herein for alkyl. An optionally substituted non-aromatic aliphatic is optionally substituted as described herein for cycloalkyl. In some embodiments, an acyclic aliphatic is alkyl. In certain embodiments, a cyclic aliphatic is aryl. In particular embodiments, a cyclic aliphatic is cycloalkyl. The term “alkanoyl,” as used herein, represents a chemical substituent of formula -C(0)-R, where R is alkyl. An optionally substituted alkanoyl is an alkanoyl that is optionally substituted as described herein for alkyl.

The term “alkoxy,” as used herein, represents a chemical substituent of formula -OR, where R is a Ci-6 alkyl group, unless otherwise specified. An optionally substituted alkoxy is an alkoxy group that is optionally substituted as defined herein for alkyl.

The term “alkyl,” as used herein, refers to an acyclic straight or branched chain saturated hydrocarbon group, which, when unsubstituted, has from 1 to 12 carbons, unless otherwise specified. In certain preferred embodiments, unsubstituted alkyl has from 1 to 6 carbons. Alkyl groups are exemplified by methyl; ethyl; n- and iso-propyl; n-, sec-, iso- and tert-butyl; neopentyl, and the like, and may be optionally substituted, valency permitting, with one, two, three, or, in the case of alkyl groups of two carbons or more, four or more substituents independently selected from the group consisting of: alkoxy; acyloxy; amino; aryl; aryloxy; azido; cycloalkyl; cycloalkoxy; halo; heterocyclyl; heteroaryl; heterocyclylalkyl; heteroarylalkyl; heterocyclyloxy; heteroaryloxy; hydroxy; nitro; thiol; silyl; cyano; =0; =S; and =NR’, where R’ is H, alkyl, aryl, or heterocyclyl. In some embodiments, two substituents combine to form a group -L-CO-R, where L is a bond or optionally substituted Ci-n alkylene, and R is hydroxyl or alkoxy. Each of the substituents may itself be unsubstituted or, valency permitting, substituted with unsubstituted substituent(s) defined herein for each respective group.

The term “alkylene,” as used herein, represents a divalent substituent that is an alkyl having one hydrogen atom replaced with a valency. An optionally substituted alkylene is an alkylene that is optionally substituted as described herein for alkyl.

The term “altmer,” as used herein, refers to an oligonucleotide having a pattern of structural features characterized by internucleoside linkages, in which no two consecutive internucleoside linkages have the same structural feature. In some embodiments, an altmer is designed such that it includes a repeating pattern. In some embodiments, an altmer is designed such that it does not include a repeating pattern. In instances, where the “same structural feature” refers to the stereochemical configuration of the internucleoside linkages, the altmer is a “stereoaltmer.”

The term “aryl,” as used herein, represents a mono-, bicyclic, or multicyclic carbocyclic ring system having one or two aromatic rings. Aryl group may include from 6 to 10 carbon atoms. All atoms within an unsubstituted carbocyclic aryl group are carbon atoms. Non-limiting examples of carbocyclic aryl groups include phenyl, naphthyl, 1 ,2-dihydronaphthyl, 1 ,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl, etc. The aryl group may be unsubstituted or substituted with one, two, three, four, or five substituents independently selected from the group consisting of: alkyl; alkoxy; acyloxy; amino; aryl; aryloxy; azido; cycloalkyl; cycloalkoxy; halo; heterocyclyl; heteroaryl; heterocyclylalkyl; heteroarylalkyl; heterocyclyloxy; heteroaryloxy; hydroxy; nitro; thiol; silyl; and cyano. Each of the substituents may itself be unsubstituted or substituted with unsubstituted substituent(s) defined herein for each respective group.

The term “aryl alkyl,” as used herein, represents an alkyl group substituted with an aryl group.

The aryl and alkyl portions may be optionally substituted as the individual groups as described herein.

The term “arylene,” as used herein, represents a divalent substituent that is an aryl having one hydrogen atom replaced with a valency. An optionally substituted arylene is an arylene that is optionally substituted as described herein for aryl. The term “aryloxy,” as used herein, represents a group -OR, where R is aryl. Aryloxy may be an optionally substituted aryloxy. An optionally substituted aryloxy is aryloxy that is optionally substituted as described herein for aryl.

The term “bicyclic sugar moiety,” as used herein, represents a modified sugar moiety including two fused rings. In certain embodiments, the bicyclic sugar moiety includes a furanosyl ring.

The term “blockmer,” as used herein, refers to an oligonucleotide strand having a pattern of structural features characterized by the presence of at least two consecutive internucleoside linkages with the same structural feature. By same structural feature is meant the same stereochemistry at the internucleoside linkage phosphorus or the same modification at the linkage phosphorus. The two or more consecutive internucleoside linkages with the same structure feature are referred to as a “block.” In instances, where the “same structural feature” refers to the stereochemical configuration of the internucleoside linkages, the blockmer is a “stereoblockmer.”

The expression “C_x-y,” as used herein, indicates that the group, the name of which immediately follows the expression, when unsubstituted, contains a total of from x to y carbon atoms. If the group is a composite group (e.g., aryl alkyl), C_x-y indicates that the portion, the name of which immediately follows the expression, when unsubstituted, contains a total of from x to y carbon atoms. For example, (Ce-io- aryl)-Ci-6-alkyl is a group, in which the aryl portion, when unsubstituted, contains a total of from 6 to 10 carbon atoms, and the alkyl portion, when unsubstituted, contains a total of from 1 to 6 carbon atoms.

The term "complementary," as used herein in reference to a nucleobase sequence, refers to the nucleobase sequence having a pattern of contiguous nucleobases that permits an oligonucleotide having the nucleobase sequence to hybridize to another oligonucleotide or nucleic acid to form a duplex structure under physiological conditions. Complementary sequences include Watson-Crick base pairs formed from natural and/or modified nucleobases. Complementary sequences can also include non- Watson-Crick base pairs, such as wobble base pairs (guanosine-uracil, hypoxanthine-uracil, hypoxanthine-adenine, and hypoxanthine-cytosine) and Hoogsteen base pairs.

The term “contiguous,” as used herein in the context of an oligonucleotide, refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to each other.

For example, “contiguous nucleobases” means nucleobases that are immediately adjacent to each other in a sequence.

The term “cycloalkyl,” as used herein, refers to a cyclic alkyl group having from three to ten carbons (e.g., a C3-C10 cycloalkyl), unless otherwise specified. Cycloalkyl groups may be monocyclic or bicyclic. Bicyclic cycloalkyl groups may be of bicyclo[p.q.0]alkyl type, in which each of p and q is, independently, 1 , 2, 3, 4, 5, 6, or 7, provided that the sum of p and q is 2, 3, 4, 5, 6, 7, or 8. Alternatively, bicyclic cycloalkyl groups may include bridged cycloalkyl structures, e.g., bicyclo[p.q.r]alkyl, in which r is 1 , 2, or 3, each of p and q is, independently, 1 , 2, 3, 4, 5, or 6, provided that the sum of p, q, and r is 3, 4, 5, 6, 7, or 8. The cycloalkyl group may be a spirocyclic group, e.g., spiro[p.q]alkyl, in which each of p and q is, independently, 2, 3, 4, 5, 6, or 7, provided that the sum of p and q is 4, 5, 6, 7, 8, or 9. Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 1- bicyclo[2.2.1 .jheptyl, 2-bicyclo[2.2.1 .jheptyl, 5-bicyclo[2.2.1 .jheptyl, 7-bicyclo[2.2.1 .jheptyl, and decalinyl. The cycloalkyl group may be unsubstituted or substituted (e.g., optionally substituted cycloalkyl) with one, two, three, four, or five substituents independently selected from the group consisting of: alkyl; alkoxy; acyloxy; amino; aryl; aryloxy; azido; cycloalkyl; cycloalkoxy; halo; heterocyclyl; heteroaryl; heterocyclylalkyl; heteroarylalkyl; heterocyclyloxy; heteroaryloxy; hydroxy; nitro; thiol; silyl; cyano; =0; =S; =NR’, where R’ is H, alkyl, aryl, or heterocyclyl. Each of the substituents may itself be unsubstituted or substituted with unsubstituted substituent(s) defined herein for each respective group.

The term “cycloalkylene,” as used herein, represents a divalent substituent that is a cycloalkyl having one hydrogen atom replaced with a valency. An optionally substituted cycloalkylene is a cycloalkylene that is optionally substituted as described herein for cycloalkyl.

The term “cycloalkoxy,” as used herein, represents a group -OR, where R is cycloalkyl. Cycloalkoxy may be an optionally substituted cycloalkoxy. An optionally substituted cycloalkoxy is cycloalkoxy that is optionally substituted as described herein for cycloalkyl.

The term “duplex,” as used herein, represents two oligonucleotides that are paired through hybridization of complementary nucleobases.

The term “gapmer,” as used herein, refers to an oligonucleotide having an RNase H recruiting region (gap) flanked by a 5' wing and 3' wing, each of the wings including at least one affinity enhancing nucleoside (e.g., 1 , 2, 3, or 4 affinity enhancing nucleosides).

The term “halo,” as used herein, represents a halogen selected from bromine, chlorine, iodine, and fluorine.

The term “headmer,” as used herein, refers to an oligonucleotide having an RNase H recruiting region (gap) flanked by a 5’ wing including at least one affinity enhancing nucleoside (e.g., 1 , 2, 3, or 4 affinity enhancing nucleosides).

The term “heteroalkyl,” as used herein refers to an alkyl group interrupted one or more times by one or two heteroatoms each time. Each heteroatom is, independently, O, N, or S. None of the heteroalkyl groups includes two contiguous oxygen atoms. The heteroalkyl group may be unsubstituted or substituted (e.g., optionally substituted heteroalkyl). When heteroalkyl is substituted and the substituent is bonded to the heteroatom, the substituent is selected according to the nature and valency of the heteratom. Thus, the substituent bonded to the heteroatom, valency permitting, is selected from the group consisting of =0, -N(R^N2)2, -SC>₂0R^N3, -SC>2R^N2, -SOR^N3, -COOR^N3, an N protecting group, alkyl, aryl, cycloalkyl, heterocyclyl, or cyano, where each R^N2 is independently H, alkyl, cycloalkyl, aryl, or heterocyclyl, and each R^N3 is independently alkyl, cycloalkyl, aryl, or heterocyclyl. Each of these substituents may itself be unsubstituted or substituted with unsubstituted substituent(s) defined herein for each respective group. When heteroalkyl is substituted and the substituent is bonded to carbon, the substituent is selected from those described for alkyl, provided that the substituent on the carbon atom bonded to the heteroatom is not Cl, Br, or I. It is understood that carbon atoms are found at the termini of a heteroalkyl group. In some embodiments, heteroalkyl is PEG

The term “heteroalkylene,” as used herein, represents a divalent substituent that is a heteroalkyl having one hydrogen atom replaced with a valency. An optionally substituted heteroalkylene is a heteroalkylene that is optionally substituted as described herein for heteroalkyl.

The term “heteroaryl,” as used herein, represents a monocyclic 5-, 6-, 7-, or 8-membered ring system, or a fused or bridging bicyclic, tricyclic, or tetracyclic ring system; the ring system contains one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and at least one of the rings is an aromatic ring. Non-limiting examples of heteroaryl groups include benzimidazolyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, furyl, imidazolyl, indolyl, isoindazolyl, isoquinolinyl, isothiazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, purinyl, pyrrolyl, pyridinyl, pyrazinyl, pyrimidinyl, qunazolinyl, quinolinyl, thiadiazolyl (e.g., 1 ,3,4-thiadiazole), thiazolyl, thienyl, triazolyl, tetrazolyl, dihydroindolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, etc. The term bicyclic, tricyclic, and tetracyclic heteroaryls include at least one ring having at least one heteroatom as described above and at least one aromatic ring. For example, a ring having at least one heteroatom may be fused to one, two, or three carbocyclic rings, e.g., an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or another monocyclic heterocyclic ring. Examples of fused heteroaryls include 1 ,2,3,5,8,8a-hexahydroindolizine; 2,3-dihydrobenzofuran; 2,3-dihydroindole; and 2,3- dihydrobenzothiophene. Heteroaryl may be optionally substituted with one, two, three, four, or five substituents independently selected from the group consisting of: alkyl; alkoxy; acyloxy; aryloxy; amino; arylalkoxy; cycloalkyl; cycloalkoxy; halogen; heterocyclyl; heterocyclyl alkyl; heteroaryl; heteroaryl alkyl; heterocyclyloxy; heteroaryloxy; hydroxyl; nitro; thiol; cyano; =0; -NR2, where each R is independently hydrogen, alkyl, acyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl; -COOR^A, where R^A is hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl; and -CON(R^B)2, where each R^B is independently hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl. Each of the substituents may itself be unsubstituted or substituted with unsubstituted substituent(s) defined herein for each respective group.

The term “heteroaryloxy,” as used herein, refers to a structure -OR, in which R is heteroaryl. Heteroaryloxy can be optionally substituted as defined for heteroaryl.

The term “heterocyclyl,” as used herein, represents a monocyclic, bicyclic, tricyclic, or tetracyclic ring system having fused or bridging 4-, 5-, 6-, 7-, or 8-membered rings, unless otherwise specified, the ring system containing one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. Heterocyclyl may be aromatic or non-aromatic. An aromatic heterocyclyl is heteroaryl as described herein. Non-aromatic 5-membered heterocyclyl has zero or one double bonds, non-aromatic 6- and 7-membered heterocyclyl groups have zero to two double bonds, and non-aromatic 8-membered heterocyclyl groups have zero to two double bonds and/or zero or one carbon- carbon triple bond. Heterocyclyl groups have a carbon count of 1 to 16 carbon atoms unless otherwise specified. Certain heterocyclyl groups may have a carbon count up to 9 carbon atoms. Non-aromatic heterocyclyl groups include pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, homopiperidinyl, piperazinyl, pyridazinyl, oxazolidinyl, isoxazolidiniyl, morpholinyl, thiomorpholinyl, thiazolidinyl, isothiazolidinyl, thiazolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyranyl, dihydropyranyl, dithiazolyl, etc. The term “heterocyclyl” also represents a heterocyclic compound having a bridged multicyclic structure in which one or more carbons and/or heteroatoms bridges two non-adjacent members of a monocyclic ring, e.g., quinuclidine, tropanes, or diaza-bicyclo[2.2.2]octane. The term “heterocyclyl” includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heterocyclic rings is fused to one, two, or three carbocyclic rings, e.g., a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or another heterocyclic ring. Examples of fused heterocyclyls include 1 ,2,3,5,8,8a-hexahydroindolizine; 2,3-dihydrobenzofuran; 2,3-dihydroindole; and 2,3-dihydrobenzothiophene. The heterocyclyl group may be unsubstituted or substituted with one, two, three, four or five substituents independently selected from the group consisting of: alkyl; alkoxy; acyloxy; aryloxy; amino; arylalkoxy; cycloalkyl; cycloalkoxy; halogen; heterocyclyl; heterocyclyl alkyl; heteroaryl; heteroaryl alkyl; heterocyclyloxy; heteroaryloxy; hydroxyl; nitro; thiol; cyano; =0; =S; -NR2, where each R is independently hydrogen, alkyl, acyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl; -COOR^A, where R^A is hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl; and -CON(R^B)2, where each R^B is independently hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl.

The term “heterocyclyl alkyl,” as used herein, represents an alkyl group substituted with a heterocyclyl group. The heterocyclyl and alkyl portions of an optionally substituted heterocyclyl alkyl are optionally substituted as described for heterocyclyl and alkyl, respectively.

The term “heterocyclylene,” as used herein, represents a divalent substituent that is a heterocyclyl having one hydrogen atom replaced with a valency. An optionally substituted heterocyclylene is a heterocyclylene that is optionally substituted as described herein for heterocyclyl.

The term “heterocyclyloxy,” as used herein, refers to a structure -OR, in which R is heterocyclyl. Heterocyclyloxy can be optionally substituted as described for heterocyclyl.

The terms “hydroxyl” and “hydroxy,” as used interchangeably herein, represent -OH.

The term “hydrophobic moiety,” as used herein, represents a monovalent group covalently linked to an oligonucleotide backbone, where the monovalent group is a bile acid (e.g., cholic acid, taurocholic acid, deoxycholic acid, oleyl lithocholic acid, or oleoyl cholenic acid), glycolipid, phospholipid, sphingolipid, isoprenoid, vitamin, saturated fatty acid, unsaturated fatty acid, fatty acid ester, triglyceride, pyrene, porphyrine, texaphyrine, adamantine, acridine, biotin, coumarin, fluorescein, rhodamine, Texas- Red, digoxygenin, dimethoxytrityl, f-butydimethylsilyl, f-butyldiphenylsilyl, cyanine dye (e.g., Cy3 or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen. Non-limiting examples of the monovalent group include ergosterol, stigmasterol, b-sitosterol, campesterol, fucosterol, saringosterol, avenasterol, coprostanol, cholesterol, vitamin A, vitamin D, vitamin E, cardiolipin, and carotenoids. The linker connecting the monovalent group to the oligonucleotide may be an optionally substituted Ci-eo aliphatic (e.g., optionally substituted Ci-eo alkylene) or an optionally substituted C2-60 heteroaliphatic (e.g., optionally substituted C2-60 heteroalkylene), where the linker may be optionally interrupted with one, two, or three instances independently selected from the group consisting of an optionally substituted arylene, optionally substituted heterocyclylene, and optionally substituted cycloalkylene. The linker may be bonded to an oligonucleotide through, e.g., an oxygen atom attached to a 5’-terminal carbon atom, a 3’-terminal carbon atom, a 5’-terminal phosphate or phosphorothioate, a 3’-terminal phosphate or phosphorothioate, or an internucleoside linkage.

The term “internucleoside linkage,” as used herein, represents a group or bond that forms a covalent linkage between adjacent nucleosides in an oligonucleotide. An internucleoside linkage is an unmodified internucleoside linkage or a modified internucleoside linkage. An “unmodified internucleoside linkage” is a phosphate (-0-P(0)(0H)-0-) internucleoside linkage (“phosphate phosphodiester”). A “modified internucleoside linkage” is an internucleoside linkage other than a phosphate phosphodiester. The two main classes of modified internucleoside linkages are defined by the presence or absence of a phosphorus atom. Non-limiting examples of phosphorus-containing internucleoside linkages include phosphodiester linkages, phosphotriester linkages, phosphorothioate diester linkages, phosphorothioate triester linkages, morpholino internucleoside linkages, methylphosphonates, and phosphoramidate. Nonlimiting examples of non-phosphorus internucleoside linkages include methylenemethylimino ( — CH2 — N(CH₃)— O— CM2— ), thiodiester (— O— C(O)— S— ), thionocarbamate (— O— C(0)(NH)— S— ), siloxane ( — O — Si(H)2 — O — ), and N,N'-dimethylhydrazine ( — CH2 — N(CH₃) — N(CH₃) — ). Phosphorothioate linkages are phosphodiester linkages and phosphotriester linkages in which one of the non-bridging oxygen atoms is replaced with a sulfur atom. In some embodiments, an internucleoside linkage is a group of the following structure:

where

Z is O, S, or Se;

Y is -X-L-R¹; each X is independently -O-, -S-, -N(-L-R¹)-, or L; each L is independently a covalent bond or a linker (e.g., optionally substituted Ci-eo aliphatic linker or optionally substituted C2-60 heteroaliphatic linker); each R¹ is independently hydrogen, -S-S-R², -O-CO-R², -S-CO-R², optionally substituted C1-9 heterocyclyl, or a hydrophobic moiety; and each R² is independently optionally substituted C1-10 alkyl, optionally substituted C2-10 heteroalkyl, optionally substituted Ce-io aryl, optionally substituted Ce-io aryl Ci-e alkyl, optionally substituted C1-9 heterocyclyl, or optionally substituted C1-9 heterocyclyl Ci-e alkyl.

When L is a covalent bond, R¹ is hydrogen, Z is oxygen, and all X groups are -O-, the internucleoside group is known as a phosphate phosphodiester. When L is a covalent bond, R¹ is hydrogen, Z is sulfur, and all X groups are -O-, the internucleoside group is known as a phosphorothioate diester. When Z is oxygen, all X groups are -O-, and either (1) L is a linker or (2) R¹ is not a hydrogen, the internucleoside group is known as a phosphotriester. When Z is sulfur, all X groups are -O-, and either (1) L is a linker or (2) R¹ is not a hydrogen, the internucleoside group is known as a phosphorothioate triester. Nonlimiting examples of phosphorothioate triester linkages and phosphotriester linkages are described in US 2017/0037399, the disclosure of which is incorporated herein by reference.

The term “morpholino,” as used herein in reference to a class of oligonucleotides, represents an oligomer of at least 10 morpholino monomer units interconnected by morpholino internucleoside linkages. A morpholino includes a 5’ group and a 3’ group. For example, a morpholino may be of the following structure:

where n is an integer of at least 10 (e.g., 12 to 30) indicating the number of morpholino units; each B is independently a nucleobase;

R¹ is a 5’ group;

R² is a 3’ group; and

L is (i) a morpholino internucleoside linkage or, (ii) if L is attached to R², a covalent bond.

A 5’ group in morpholino may be, e.g., hydroxyl, a hydrophobic moiety, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, disphorodithioate, triphosphorodithioate, phosphonate, phosphoramidate, a cell penetrating peptide, an endosomal escape moiety, or a neutral organic polymer. A 3’ group in morpholino may be, e.g., hydrogen, a hydrophobic moiety, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, disphorodithioate, triphosphorodithioate, phosphonate, phosphoramidate, a cell penetrating peptide, an endosomal escape moiety, or a neutral organic polymer.

The term “morpholino internucleoside linkage,” as used herein, represents a divalent group of the following structure:

Z

-I_c1_ _c2_

⁵ J, where

Z is O or S;

X¹ is a bond, -CH2-, or -O-;

X² is a bond, -CH2-O-, or -O-; and

Y is -NR2, where each R is independently C1-6 alkyl (e.g., methyl), or both R combine together with the nitrogen atom to which they are attached to form a C2-9 heterocyclyl (e.g., N-piperazinyl); provided that both X¹ and X² are not simultaneously a bond.

The term “NRL,” as used herein, represents refers to a ribonucleic acid (e.g., pre-mRNA or mRNA) that encodes the protein Neural Retina Leucine Zipper in humans. An exemplary genomic DNA sequence of a human NRL gene is given by SEQ ID NO. 1 (NCBI Reference Sequence: NG_011697.2). One of skill in the art will recognize that a pre-mRNA is produced from the genomic DNA in accordance with the central dogma; pre-mRNA is then spliced to produce transcripts, e.g., NRL transcript 1 , NRL transcript 2, NRL transcript 3, or NRL transcript 4. Exemplary mRNA sequences of a human NRL gene are given by SEQ ID NOs. 2, 3, 4, and 5 (NCBI Reference Sequences: NM_006177.4, NM_001354768.1 , NM_001354769.1 , and NM_001354770.1). SEQ ID NO. 2 corresponds to NRL transcript 1 . SEQ ID NO. 3 corresponds to NRL transcript 2. SEQ ID NO. 4 corresponds to NRL transcript 3. SEQ ID NO. 5 corresponds to NRL transcript 4. SEQ ID NOs. 2, 3, 4, and 5 are based on NCBI Reference Sequences for NRL transcripts 1 , 2, 3, and 4, which are provided as RNA sequences with thymidines in the NCBI Reference Sequences. One of skill in the art will recognize that an RNA sequence typically includes uridines instead of thymidines. Accordingly, target RNA sequences may include one or more uridines instead of thymidines without affecting the sequence of an oligonucleotide of the invention.

SEQ ID NO: 1 has the following nucleobase sequence (5’-3’)

1 tcttacaaac cttggttaat aaggagcttg tcaaattccc acaccagtcc gctttcatgc 61 cctcctgggc aactagaacc ttagcggcct ggctgtacag tcctgacttc tggcccacct

121 tgctgtcaat gaaggtgatg ccatcctggt gtccagccag tgcacccaca ggcttggggt

181 catcctcccg catggtgcgt cgatcccaca ctttgcagat ggcatcatct cccccagaga

241 acaggatttg ggagcttata tcagcaaagg ccactgcatt cacatcatcc tcatgggact

301 caatctaggc gaaaaggagg ccaggatata agacctcttt ctaggtctta taaaaagcca

361 aaaaccagga ggcagaggct ctatctcagg agccatacct gaagggtgcg ccggttctgt

421 tctcggtcaa agacatacag gcagccatca ttggccctga aagggaaggg ataaaggagg

481 gtcctggagc catgatcgag aggaccaacg tggtatatgc tctagagagt tccactgaga

541 aatcctgctc tgggtcaagc cctttgctct ttttatacag gaagtggggg aagagttagc 601 tgtgtttcaa ggaggcttga cacctccaat ctcgtgctgg ggaaatggag agcaaggggt

661 gaagcaactt gtaaatcctg accttgggag aaggtctggt tgttctctgc agctgccagt

721 gccatctgtg taacagcaga actcatctct ggcagaaccc ccatcttgtt attgagggaa

781 acatacccca caagcactta ccctcctagt acttctcgtc catctgagga gacagcaatg

841 gagaagacag caaagcgacg ctcatctggc ctaaaagcaa gaaggagctg ggttaagtgg

901 acttccttca gcttttgtta atctacataa aatctgacaa cggttgtaag gcacatcccc

961 aggggaaaaa agaagtctgg ctaacatgca ccataatgca gatgctcagt gagaacccag

1021 aaatcatcct gcaaccatct attttaccca gtcctctggc tgacaagatg gtgcttccct

1081 cccaggaatg cacacagacc caccagtata ctttttcccc taaacagaac ttccaactca

1141 agaggtttgc tcccaaagag cttcccttga gaagtttctg atgagtctga ccagaaaggg

1201 aagccagtac ctgagatcca gggcagtgtg tgtatctccc tcaccataga tattgcagat

1261 atgaactaag agaagcaaga aaaacaagaa tctttcaggg atgggggaag aagagtggaa

1321 gtgaagctag ggaagagatc aaacagggga caagggtact ggagcacttt gggagtgatt

1381 ttcccaggac aaagtgaggg atcctggctc agaggggaca ggcacatgga aacctcaggt

1441 cccaggctgg agagtcagtg gaagcctggt gcatactcac tgtaatcaga ccagctagag

1501 tagaggaagt ggttcccatc aggggtgaag gccacatcca agacgctcca gcctacgtcg

1561 cgggccttga tgctcttgaa tttacggaaa cggccatatc ggcagtcata gagtcggatt

1621 gtctggtctg tgtaagtaaa gatcaggcag aattatggga cagtttcctt gtaacttttc

1681 tagattagac atctcccact cagtgtacct gtggagtctt ccctttatca tgggaaacat

1741 ggtctggcta agcatcctta agccaggtct gagctccccg tctgtggctt tctagcctca

1801 gtcaggggga atgtcccatg ttccaggaaa agatagttta taggaccagg gtctggtacc

1861 ttggcaagca gacatgaata tttgaccatc tttgctgtag atgccacaga aagccttctg

1921 agagtagcta tcagtgaagc ccagatcatt gggcaagaag ctagggaagg agagagaaag

1981 tgagggtgag gggtgctcag gataaggagt gtcccaatca gcagaatcct aacagagctg

2041 gaatatcaga acagtcattc ttcccttttg gctgtgagtg caatcccgtc tttcaaccat

2101 tccaccatcc tctaaatttc tacaattttc tacacttttc tactttgttt actctcaaag

2161 tttcaacccc atttaaccta acctttaata tctctgaggc tcctttaaca cctacccctt

2221 caaagtttag gagataagac ctatctggcc cttagagtct cttcaccaag ccccatactc

2281 actgagatat cactcgagac tgttctccaa gggagaagct tccccgatgg cagaggcccc

2341 gttctctcta aatggaaggg attggggaat attgctagca gaaatagagg gccacttcag

2401 ccatatcaag ccccctcctc caaagatatt gctcttgtta tagaaatgga cacacacatc

2461 atagtctgtc ccacagaact ccgatggagg ctggttaact ctccagcccc agcgcttgag

2521 gcttctactt tggaaagttg acttcctttt tctctggggc tggcagagct tctggctgct

2581 ttggcaggcc tgctgccagg ctgggaggtg gagaggccta cctggtgcaa cattcgagga

2641 aagctgtgct tctgggcggc ccgcctaagc cccagctgcc ctgtggccag ttccacttgt

2701 gtcttgatct cattgaattc cagctcccgg gtgtcagggg tagcatccac tgtgagtacc

2761 aaggagaagt ttcatcctag gtctgccagc ctcagaggac agttagcaga actgtaccca

2821 agcccacccc tgtggctgaa tatactgggg ctgtaaggac tccttattcc ttaaattatc

2881 gtaccttctt tcaatgtaac ctatcattca aaatatcact aaaaggtttt ctagtaaaag

2941 tcttccaaca ttaggggctt ttcctcttac caggtgggtt gtatcgatcc ccaagacgac

3001 catcccaagc tctgtcattc tcttcctctg agtccaagag ggcctgaatg aattgtaaat

3061 ttgctgcacc tcctccctgc accaacctca cttggcctct gtggagagac ccccattata

3121 aaggagggga cttcttcccc caaaccaacc aaattcagag tcaatggcta tctagctctt 3181 agaccacttg gtactcattt tgaggtttga ggctgactag accttggctg ttatttttgg

3241 tctcctataa aacacagtag gaaagagagt tttactctta cagcaagagc aagaccaaca

3301 tgctgtggac gtgacctact aagttgacct cccagcgagc taaacattct aacatcattt

3361 ccttagaaca agagcaagac caatacgctg tggacatgac ctactaagtt gacctcccaa

3421 caagctaaac atcctcacag catttccttc tgagcctctg cctgagaagt ccaccacaga

3481 agacaatatt ctaagctggg tgcggtggtg tgtgcaactg tagtcttagc tacctgggag

3541 gcaaggattg cttgagccca ggagttgcag gctgtggtgt gcacttgatc acacctgtga

3601 atagcccgtt ctccagactg gggaacacag tgagccctgt ctatgtctac taaaaacaac

3661 acaaaacaca aacaaaaaag aatacaatat tctgaaggga cccccaaacc catcttctcc

3721 tcaaagagcc ataagcaaag aaagaaagcc agctgcaccc ttttccagtc aattccaggt

3781 ggtctctccc ttcaaggcat tctttcctca agaacaggtt agagtcaggg caaattaaac

3841 ttgtttttca agtaaatcga agtggcctca tatcatgtgt caactgcctt actaaaaagt

3901 tcaacactcc cttcccatct cttcacccct tacatcactc ctttcccact aggacccccc

3961 cctttttttt ttcaagctta cttggaaaat agactatatt tgtgtagtca cagagacagg

4021 ctcccttgaa tagcccctca aacacatgag caggtcctct tggtgaccct ccctaggtaa

4081 gaaccactct tactgggtat ggagctgagt gccatgggtg cccagcaggg aaaggctgtt

4141 cagctcaaac agaacagctg tcctgagcga atcccatttc ctaacctctg taattttcct

4201 gcctggagtt gggatcaagg cactgtggta gcacctgctg ggggtcacac cagagggtaa

4261 gttacctgcg gaggagatag gccagtacct gggccagatc cacatcttca tcctcttctt

4321 cctcttcctc actccgacgc aggccagccc ctcttcgggg caagccctcg gaggggtctc

4381 cggacccgga tcctgcactg ctgctgttcc gcgatcccat cttctggtca cagcattctt

4441 aggtcctgtg cgggggctcc tcctgtcacc tccttgggtt tggtgaaagc aaagcgtgct

4501 ctagaaatac cagggtctcc tgagacgggg tagtagctat ctctaggagg gtgacccttc

4561 ctcggggctg agatcccttt tctttagcct atgccaacta ccaaggcgtc gggaacacaa

4621 accttctcca atcctgtgaa gcaaagaaag caagagtgaa ccgtagtgcg ctaccctagc

4681 tcaccaactg aagaaagatt tgttttctac accaaactca cctatcgaag tccccggact

4741 ggggagcggg aggggtgttg ggcggtgttg gatttccagg taacctctct gaccgaccaa

4801 tcgaaatccc tctcggaaga aagcgcttca ctagcttatc tcatctaacc aattagaagc

4861 cttaggactc aaagaccctc gacgccccca ccttatcgac caatcagagc gcccccttac

4921 aaaggccggc agcagtgaca gccaatgaaa atagaatttg gcctaacacc cctccttcct

4981 cgcctcacgc ccacgtggtg cgccgggggt gtgtacgcca tgagggggag gaagccgaga

5041 agcacctccc actgaagccg gcggtaaccg ccccacgcga gaccacccac cagccaggag

5101 cggtcgcagg acccgcagcg gcgcgaggcc ttgtttacac cgactgccgc cactgcgttg

5161 gccaaccgct cctccgtcac gcgcttcgct cgtcacgcat ctcgcggctt cctggccggc

5221 gggctgcaaa cgtcacacga cggccccgcc cccttggcca gccaactgga cagcggagca

5281 cgatgattga taggtaggcg ggagaatgcg cgaggtgcct gaggggagga gaaagtgcac

5341 acccaaggag ggctgttagc tcttgctggg gaggagtaag tgggaactgc cagcttcctg

5401 aatcagagac agccgaacta ggcgtaacgg ctgagaggcg gggcttaaaa aagaaacgtt

5461 tggtggttcg ctaccgagag gtattttagc aagggatata atttactgtc tgtccctaaa

5521 gatcatttgg catcgcactt ttcagacagt gcccaagggc ctcctttctc cccactgctt

5581 cccctgtcca gctggagccg ttcttctgtc gctggccgca gctccagtcg gttctgccct

5641 aaggggtgcc cagcgtccca gtagacctcc ataccgtcct tccgggctta gcttttggaa

5701 gccttgctct tgggacggtc cccttttaga cctcccttta tccggtctga caatttttta 5761 agacaatccc tggctttccg ccgcggtctt tgcagacgtt ccctaataca cgcgtctttc

5821 tgtcaaagct tttccggttc cgctgtcccc tttgacgctc ccttggccct ccgggatttg

5881 cagaccttcg ctagtccctc tgtcctcgcg gtgcctctcc gcccccggcc gcgggtccag

5941 ggtgcgcgct cgccgcctcc tgccccctcc tgccccctcc tgcgggttta tgagtaacaa

6001 gcctgagtcc aaggccggga gttgccacat ctctgatcct cgccctggct gggtggggac

6061 gtgtgggttg aatgaccttg ctgtctcggg ctcagaggct cagggtccga gctgaacttc

6121 attccctgca gcctgcactt ccttgggaaa gctgcgagct tccctccgcc cgctcctgag

6181 ggcagggatg ggagcgaggg ctcgtggcct tcttgggccc tcctggggcc tctcccagtg

6241 agggcgaggg tttggccagc tttcgggcga cgtctcctag tctctagatc cggagacctg

6301 agcttgtcac cagggctagc tttgatggcc tctgcgctta gaagggtcct gaatgtttgg

6361 tttaatgttc tagtgttaac ggtcttgaaa ttattagtaa ttttaaagca agtgcctggt

6421 attttcatct tgcactcggc tccactgatt atgtaggcgg tctgctgggc gcccatcctc

6481 tattgccaga ttacttacta tctgtgcttt aacgtttatt gagactttag gattccgtgg

6541 tttctgggtt atccagcctg gaaaatcggg atctttattt attgattgat tttacctgcg

6601 ttctcccaga atgtgacgga atcatttttt ttttttatta tactctaagt tttagggtac

6661 atgtgcacat tgtgcaggtt agttacatat gtatacatgt gccatgctgg tgcgctgcac

6721 cactaacgtg tcatctagca ttaggtatat ctcccaatgc tatccctccc ccctcccccg

6781 accccaccac agtccccaga gtgtgatatt ccccttcctg tgtccatgtg atctcattgt

6841 tcaattccca cctatgagtg agaatatgcg gtgtttggtt ttttgttctt gcgatagttt

6901 actgagaatg atggtttcca atttcatcca tgtccctaca aaggacatga actcatcatt

6961 ttttatggct gcatagtatt ccatggtgta tatgtgccac attttcttaa tccagtctat

7021 cattgttgga catttgggtt ggttccaagt ctttgctatt gtgaatagtg ccgcaataaa

7081 catacgtgtg catgtgtctt tatagcagca tgatttatag tcctttgggt atatacccag

7141 taatgggatg gctgggtcaa atggtatttc tagttctaga tccctgagga atcgccacac

7201 tgacttccac aatggttgaa ctagtttaca gtcccaccaa cagtgtaaaa gtgttcctat

7261 ttctccacat cctctccagc acctgttgtt tcctgacttt ttaatgattg ccattctaac

7321 tggtgtgaga tgatatctca tagtggtttt gatttgcatt tctctgatgg ccagtgatga

7381 tgagcatttc ttcatgtgtt ttttggctgc ataaatgtct gcttttgaga agtgtctgtt

7441 catgtccttc gcccactttt tgatggggtt gtttgttttt ttcttgtaaa tttgtttgag

7501 ttcattgtag attctggata ttagcccttt gtcagatgag taggttgcga aaattttctc

7561 ccatgttgta ggttgcctgt tcactctgat ggtagtttct tttgctgtgc agaagctctt

7621 tagtttaatt agatcccatt tgtcatcctc cctaactcat tttatgaggc cagcatcatt

7681 ctgataccaa agccgggcag agacacaacc aaaaaagaga attttagacc aatatccttg

7741 atgaacattg atgcaaaaat cctcaataaa atactggcaa accgaatcca gcagcacatc

7801 aaaaagctta tcgaccatga tcaagtgggc ttcatccctg ggatgcaagg ctggttcaat

7861 atacgcaaat caataaatgt aatccagcat ataaacagag ccaaagacaa aaaccacatg

7921 attatctcaa tagatgcaga aaaagccttt gacaaaattc aacaaccctt catgctaaaa

7981 actctcaata aattaggtat tgatgggacg tatttcaaaa taataagagc tatctatgac

8041 aaacccacag ccaatatcat actgaatggg caaaaactgg aagcattccc tttgaaaact

8101 ggcacaagac agggatgccc tctctcaccg ctcctattca acatagtgtt ggaagttctg

8161 gccagggcaa tcaggcagga gaaggaaata aagggtattc aattaggaaa agaggaagtc

8221 aaattgtccc tgtttgcaga cgacatgatt gtttatctag aaaaccccat cgtctcagcc

8281 caaaatctcc ttaagctgat aagcaacttc agcaaagtct caggacggaa tcatttttta 8341 aaaaaattgg ccgggcgcgg tggctcacgc ctgtaatccc agcactttgg gaggccaagg

8401 cgggtggatc acctgaggtc aggagttcga gaccagcctg gccaacatgg tgaaaccccg

8461 tcactaaaaa aaaaaaagcc gagtgtggtg gcatgcgctt gtaatcccag ccactcgaga

8521 ggctgaggca agagaatcgc ttgaacccgg gaggtggagg ttgcattgag ccgagatcac

8581 gccactgcac tccagcctgg gctacaaaag cgaaactccg tcacacacac acaaaaaaca

8641 atcaaccaac caaacaaaaa acacctatgc ctcttccggt agttgatctg ctgaagaatc

8701 ctcctctaac agcatttagt gcttaacgca caagtgagct gggcacggtg gcttacgact

8761 gtaattccag cactgtggga ggccgaggag gaggatcatt tgtggccagg agttcaaggc

8821 cagcctgggc aacatagtga gacccagtct acaaaaagaa aaaaaaaata gccggccatc

8881 gtggcgcgtg cctatggtcc caacttactt gggaaggatg aaggagggaa tcttgagcct

8941 atgaggtgga ggctgcagtg agccatgatt gctccactgt actccagcct ggggatagag

9001 ctaaagactg tctctaaaag aaaatcaaca aatggctcac tattgttcat tgtttcaaat

9061 acaaacttcc aaacctggat gtctattttt agtaacctca ttttttgata tttctgctaa

9121 gcaaatatgc caataaatag cttgtttatt tttacctctg caccttccca ggagaatttc

9181 ttattagagg gaagctgtct acaaaactcc cttcctcaca gccttgcact ttgacccagt

9241 aatactgctt ccagaaatct aggccggaca tgcataaaaa tttatgttct acatagcaat

9301 tattgataaa agtggaacac tggaaacaaa gatctaaata tcttgcaata tgggattgtt

9361 aaataaatta tggtccatgt agtatagaca cagaaatacc aggtagctat taaaatggtg

9421 ttgtagtctt caattttttt ttaaggtacc tattcctggt ataggtttgt tgtagccttc

9481 tatttttata ccataggaaa ttgtgcaaac tacattgtca agttaaaaaa ataaagataa

9541 tcatttcttc tggtgacatg taatagatgc aaaaacgatg tggtttcatt ttttcattag

9601 aaagtataat aggcgtggta gctcacacct gtaatcccag tactttgaga ggctgaggcg

9661 ggcagctcgc ttgaccacag gaatttgaga tcagcctggg caatgtgacg aaaccccatc

9721 tctacgaaaa atacaaaaac aaaattgagc tgggcctgta gtcttggcta cccaggatgc

9781 tgaggtggaa ggatcacctg agtctggtca aggctgcagt gagccatgat tgcgccactg

9841 cacttcatcc agcctgggcg agaatgagac cccgtctcaa agaaaaaaaa aaaaagtata

9901 ataataccca gctcattaca tcattaaaca aagtggggag ggcttgcatg aactggtgac

9961 aaaggtgtag cttctacaaa ggattatgat gaatacagct atagcacctg aggtccagaa

10021 aaacgaacaa aaaacttctg ttaaacttat ctccttaaca attcattttt tgattctctt

10081 ttctcttctt cttacaaatt agtttaaacc tgaataaaat cttttaattc aagcttttgt

10141 aatatggaac ttgtacatta ttgagtaaaa catttaaaag tcatggtaaa tgttaaaata

10201 actgaggttt tatgacaaaa ttgagtcctt cacatcacat gggaaagaat ataagattaa

10261 tcactggatt cattgattaa tttttttgcg tataaaagat tttttttttt tttttttgag

10321 atggagtctt gctctgtcac ccaggctgga gtgcagtgga ccgatctcgg ctcactgcaa

10381 gctccgcctc ccgggttcat gctattctcc tgcctcagcc tcccgagtac ctgggactac

10441 aggcgcccgc caccatgccc ggctaatttt ttgtattttt cttagtagag acggggtttc

10501 accatgttag ccaggatggt ctcgatctcc tgacctcatg atctgaccac ctaggcctcc

10561 caaagtgctg ggattacagg tgtgagccac cacgcctggc agcatataaa agatttttaa

10621 gcctatgaac ttgattatct cactatgacc tgtaatgttt ttcaaaatgt gtaagagcat

10681 ttatatctta gggtatacag tgtgtctatt gaaataaata aaatggcata tctttaaaaa

10741 taattatggt gaagtcattt tgatagacta aattttgcct atgctgtctt gattttgaaa

10801 tttggtacgt tgcgtggtta cagtttgtga agcacttttc atttgggtaa cctctactct

10861 ctccagttgc acttaaagtc acttaaagcc acagagaggt ttttctacct tcagcaccca 10921 ttttaggaca atttctggta attgctaagt aaatgtcagc tagttgactt cctgcctgac

10981 ctgtcttggc ctgtttcctg ctcattagca tctccactag agggcaggct gggctgctga

11041 ggagagaagg aaaggagtgt tttcattctc tagctaaccc tgtcctcctc tcctctactg

11101 cagctcccag cttggtgacc acacagagct cctgggacag ttccgtgttt ggagtgactg

11161 atgctcatac atgtgtttga acattgcctt atagtattct ctgtgtcgat ggaaatggag

11221 agtgtgctaa taatttacct agggacttaa aatgtgttga ctagaaagcc actaagtaac

11281 tatgggatgt tctaatactc tcatgtcttg tgtctgtaag aatcagattt ttctgtgtgt

11341 agggtaggag ctatagatgt aatataagta aatacccttt agtggcccgg ggtagtggct

11401 cacacctgta atcccagcac tttgagaggc tgaggcaggt ggattgcttg aaaccaggag

11461 ttcaagacca gcctgggcaa catagtgaga ccccatctct aaaaaaaaaa aaaaaattag

11521 ccaggtgtgg tggcttacac ctgtagctac tcgggaggct gaggcaagag gatcatttga

11581 gcccaggaat tcaaggctgc agtgagctgt gatcacgcta ctgcattaca gcctgggcaa

11641 caaagcaaga ccatgtttcc agaaaaccct ttagccctga ttttgaatag aacatgtcag

11701 tatgagttca tgaggtttta tcttctgtcg ctatccacca aaaagatcta gaaacaatga

11761 ccaatccagt atcaatgaac actcctagca tccagattgt ggtcttaaaa cgccatttcc

11821 ccctgaaaga agccagagct tcttagagaa aaagctgatt ccaggtttgg ggaaggaaat

11881 gaacacaatg agcatggagc aacttatacc agatagccgg gaagcaagga gtatgtatca

11941 tgtcaaaagg actcagctct ttttggccta gcttccatag ccaaatgtgg gacaatttga

12001 acatttaata aggtcaattt aataaacact aagtatgttt aaatccatga gttcataatg

12061 tttgtgtgta actaactggt taccttttca aaggataata ggaaatcaaa tcattttttt

12121 gaagatgata aataaagata aagaattaag cagttatcct gctgtttcta tataaacaat

12181 accatagggt aatgaaatag aagtgtccat tttataaaat tattgcaatg ttatgacaac

12241 tcaaatatgg ctgttttgca atccctagtg agttaatgca tttagacact gaacatctga

12301 gtctggattc gtcaatgggt aaattgaagg aaaagaaagg aatggagggg gaatctgtag

12361 actgatagag acttgaaaga catatgaaaa aaattttaaa tgggagcaaa gctaaactat

12421 agtgtctaag gatctacatt taggtgatca aactataaaa aaatgcaaag aagtaattat

12481 tactcaccat agtatccact atggcaggaa ggggtggcta tgatagagat gggcaacagg

12541 gtcggggcgg ggcggggctg ggttggtgtc tgcaaagttc tgtttctttc cctgggtagt

12601 gttccaaagg tgttcacttt atgatagttt attcagccat aactttgttt tagatggtct

12661 tctgtatctg tgttttattt taaaataaaa agattataat gtgttaacta tattttatca

12721 gttatctgga gctgaagggc atgcagcccc attttattct aattaataaa aataattttt

12781 aaggctaaca ctttcttgtc cctgtgcatt tagaagatac cagaaaggtg cctggaaccc

12841 cagttccctg cctcttagct ggagggattt atttttaaat ctattttctt tctctcctag

12901 attttattta cattgctcat gttcctttta aactgtagag tcagcaatat gtaatatacg

12961 cttacagggc ctactctaat cattataaag tggaaaggtg atgatagcta acatgtatta

13021 aatggttatt cggtgccagg catagttctg agtgctttat tccactgact cttttaatct

13081 tcccaacaag actgtaaagt gtaggcattt ccattttaca gatgaagcaa ctgagaggca

13141 cctctggctc aagtctacta tgtgtttaat acctgtgcta tgttgctttt gacggtattc

13201 agttatcata tgctatttat gaagctaagg atttggctgt gttttattat tatttttaaa

13261 tttttaaatt tatttattta tttatttttt ttttgagaca gagtctcact ctgtcgccta

13321 ggctggagtg cagtggtgtg atctcagctc actgcaacct cagcctccca gtttcaagtg

13381 attctcccgc ctcagcttcc tgagtagctg gaattacagg catgggccac catgcctggc

13441 taatttttgt atttttagta gagatggggt ttcaccatgt tggtctagct ggtctcaaac 13501 tgctgacctc aggtgatctg cccacctcgg cctcccaaag tgtgagccac cgcgcctggc

13561 tggctgtgtt ttaaagtaca atatactcca ctgctaactt tgttctaacc tctgttccac

13621 tctgaccttt agatttttac acattctggc tggtcactgg catttcctat gagaacactt

13681 ggtgtttcct cttctagttt tttcctttga gggagttccc aataattcca ttttatttgt

13741 tgaagtttgt tccctttttc tgggtaaaga ctaccacact acccacctta ggttatacat

13801 tctctcagcc tgatccagat tgcacagaag ttatcctaag ctgatgcccc tgactcatgt

13861 gagatcattg cactgttttc ctgattactc tccaaagttt atttcagatg aatttaggct

13921 actctaaact gtagttcgca gaccggcagc atcagcatta cccaggagct tgtaaggaaa

13981 acaaaatctt gaggctacta cagacttgct gagtgacaat ctgcatttta acaagatctc

14041 caggtgattc aaaggcactt ttaagtttga gaaacacgga ataggtgact ttttattttt

14101 ttattttttt gagacagagt cttgctctgt cccccaggct ggagtgcaat ggcatgatct

14161 aggcttactg caacctctgc ctcctgagtt caagtgattg tcctaaatag ctgggattac

14221 aggcacctgc catcatgccc agctaatttt tgtatttttg tagagaaggg gtttcaccgt

14281 gttggccacg ctggtcttga actcccaacc tcaggtgatc cgccctcctt ggcctcccaa

14341 agtgctggga ttataggcat gagccactgc gcccggtgga ataggtgact tctaaggtcc

14401 ttgcagttgt agatgtctat actactatga aagacatgtc tttgcagttt tatcacttta

14461 acccttagct ctattgttta ctaactgtga agctttggac aaattacttg acctctctgt

14521 gcctttgtta cttcatctgt aaaatggtaa ttcccccaaa gggttgttag gtatgtgctt

14581 agaacactgc cttgttggga ataggccccc aaaatctggc cataaactgg ccccaaaact

14641 ggccataagc aaaatctctg cagcactgtg acatgtttgt gatggccatg acacccacac

14701 tggaaggttg tgggttcacc ggaatgaggg caaggaacac atggcccacc ctgggcagaa

14761 aaccgcttaa aggtattctt aaaccataaa caatagcatg agcgatctgt gccttaagga

14821 catgctcctg ctgcagataa ccagccagag cccatctctt cattttggcc catccctttg

14881 tttcccgtaa ggaatacttt tagttaatct ataaaaacaa tgcttatcac tgacttgctg

14941 tcaataaaca cgtgggtaaa tctctgttcg aggctgtcag ctctgaaggc tgtgagaccc

15001 ctgatttccc actccacacc tctatatttc tgtgtgcgtg tttttaattc ctctagcact

15061 gctgggttag ggtctccttg acagagctgg tctcagcagt gcctggcata aagaaggtgc

15121 cacagaagtg ttgttagctg tcctaatatt gaatatgctt ccatgaatac tatgcagctc

15181 ccagtgccag gtcaggcagg gggaagaaca aattcgagaa ctgacaatga agccacttca

15241 ctggagaatg gagttgtaga ggcttctgca atctaagtgg gagatcagga attttaggct

15301 tgggggctgt actgagcagc ttgctgaggc cctcagtttt ttttttgttt tgttttgttt

15361 tgtttttttt ttttgtgacg gagtttcgct ctgttgccca gactggagtg tgcagtggca

15421 cgaccttggt ttactgcaac ctctgcctcc tgggttcaca ccattctcct gcctcagctt

15481 cccaaatagc taggactaca ggcgcccgcc accactcccg gttaattttt tttttttttt

15541 tgtattttta gtagcgatag ggtttcaccg tgttagcccg gatggtctca atcccctgac

15601 ctcgtgatct gtgcgcctca gcctcccaaa gtgctgggat tacaggcgtg aaccaccaca

15661 cccagcctca ccattttttt ttttttaaga actcaggaca tagagtggat agtgccttct

15721 cccagcaatt cctatgtctg tgacccagtg ctagagctac ctgtaaaaca gatctttgat

15781 tgtgccaggc tgtgcttctt ctaggggctg gaagaagatg attgtgagac aggtgttaga

15841 gggaagcctg tgaagatagg tgggaaatga aaacagcaca aatcttgtgt aaaatagata

15901 agcatctctt attattggac agtctttgtg ggaaggtcta tggcacattg aagatgttag

15961 ttaatatcaa attgctcata ttttctgcaa ggtgccttcc ctattcccag atgagggtgc

16021 tatgtcctct tgctagacta gggcctcagg tcacattttg tgcacacgtc tctccagggc 16081 ctcaagctca gccaacacct ctttgggcag atcctggttg acctgctctg tcaggtagct

16141 ccgaatgtca cgaacctcct gttcccagaa gtccttgggg agggagaaca gctgagtggt

16201 gtctatagct ctgaggccgc tgagatccaa ggctccttcc tttggcacca gcccaatggg

16261 tgtctctcgg gcactgtcct ccccctctaa ccgccggcag atccagtcta gcacccgagc

16321 attctcccca aagcctggcc acaggaagtg ccctgcctcg tcacgccgga accagttgac

16381 atggaagata cggggcagct gggccccctt gcgcccttcc atgctcagcc agtgttccag

16441 gtagtgcccg aagttgtagc caaaaaaggg ccgcatggca aatgggtcgt gcatgatgat

16501 cttccctggg ggggaagcaa gatgggtaag gaatcttcct tctcactggg gtagggaagg

16561 gcaccctgca tccagttgga ggaagctgtg aaaggcttat ggttctgaat atcttcaggg

16621 atggggaaaa gatagaagtg ggaagaggtg gggccatatc agactccagg ggtggacaaa

16681 aggaaggtct ggctcaggaa gggagagagg acgtgctgtg tgcacacaca ggagagggag

16741 gaatggtgag ggtgctcacc tttgtgttct gctgcagcag tggactcaga gcgcatggcg

16801 ctgcccacaa acaccccatg acgccagttg aaggcctcgt ataccagggg tacccctaga

16861 tcaccaatga acagagtcac aggcagccct tttctttgga ccctaactcc agcccttctg

16921 gtgagcttga ctggtactct ctctcctaag acctcggcta tcccattttc agaccctttg

16981 cctcctccca agtttgcatt caccagggga gaactctgga gaaaggtgag atccaggctg

17041 aggctctgga tcagagggag ggaggtaggc cctaatacag agatagccct tttgccaaga

17101 acatggagct catatgttgt ttacctttgg gtctgcggcc accaaagatg atggcgtcaa

17161 tggggacacc ctctggggcc tcccaggctg ggtccatgat ggggcactgg cgagccgggg

17221 cacaaaatcg agagttggga tgtgcacagg gctccttgtc acctggcaga ggaaatacaa

17281 acactattat ttccaaggtc atgtcgaccc ccaacacaca catacatggg cacacattct

17341 tgcagggttc tttttttttt tttgcctcat cagctgccta gaaccttgtg tgataaggag

17401 cagagagagg ggagccaggg catctccaag agctcaggtt gtttagccat gaaggtgcgg

17461 ccagctgagc tatgtgagaa aagagtggga gaggaattgg ggtttggcag ggcactaatg

17521 tttgtgccaa gctactggca cttggcactg accagggctc aagggaatgg ggttagatat

17581 gggcataagg ttataataat ttttccccca gaaactattt cattatttgt gagattatta

17641 tttaaatgaa tacccaaacc taaagcaaaa actgagaatg tcagcttagt acttcagaca

17701 attgactttc cgtcagcaaa ccggggaaga caggttctcc taagttatcc aaatgcaaat

17761 aggaatttat tttatctttt tatttttgag gcagagtctt gctctgttgc ccaggctgta

17821 gtgcagtggt gcaatcttgg cttactgcaa cctccgcctc ccaggttcaa acaattctcc

17881 tacctcagcc tccccagtag ctgggattac aggcacccac caccatgcct ggctgatttt

17941 tttgtatttt taatagagat ggggtttcac catgttggcc aggctggttt caaactcctg

18001 accttaactg atctgcccac cttggcctcc caaagtagta ggattacagg catgagccac

18061 tgcgcctggc ctgattttat ttacttaatt aatttagtta tgagacggag tctcactctg

18121 tcaccaggct ggagtgcagt ggcatgatct tgtctcactg caacctctgc ctcctgggat

18181 caagcaattt tcctgcctcc tgagtagctg ggattacagg catgagccac catgcctggc

18241 taatttttgt attttattgg agacggggtt ttgccatgtt gccaggctgg tctcgaactc

18301 ctgacctcag gtgatccaac tgcctcggcc tcccaaagtg ctgggattac agatgtgagt

18361 cactgtgccc tgcaaggaat tgattttaaa gctaacaaca tgttggtgat ttccctgccc

18421 tgcttagacc ctgatgaagt gatagggctg gaaaaataga gatagggata tcacatgggt

18481 agggataacc tcaggagccc aggtcacact tggcaccttc aggtgtcctg gaggggggtt

18541 gggactaagt cagtggcaca tgtatgaagc accactgtgt ggcccctgag tggacatggt

18601 gccttctctg ctggaggttg ctggtcagac ctggggagtg gggaagccac ggggccaaaa 18661 tccccaccct ctcccatccg ccagcttctg cccgccattg cagataagag ccatccttct

18721 tcagcaccca gtgcagaatc ctaacacagg gtattattct gggaattgaa gctggggaca

18781 agatgcccta gttatgcccc caaatctgga gatgatgaaa gggttgggga actagcctgt

18841 ggggaaagat gaaagtttct tggatttctc agttgggtgg gaaggctgag gggggcttaa

18901 tgatggtgtt agtaaaaata aagggtgatt agatgaagca aggagggtga ctgctaggcc

18961 agcctggcaa acctgaatat aggcttcagt gactacaagg ccagcctccc ctcccatgct

19021 cttctttgcc cctgcctctt ggtgtctgga ctgcctgact gcagaaggcc atggaaagaa

19081 tactgaaggt ccagagtgca gggctgaggg agaggtggga ctcccaggcg cccagggtaa

19141 tgacaggatt cagaaactag cttctgggaa agactcaagc actgacctga ttttgtttgt

19201 aggaaggatt ggggtgaatg ctttggaggg acaggggaag ggtgacggta acaatactga

19261 cctttagaca gtggttatta tacaggggac agtgggagcc aagtggcagg aggagagttg

19321 gagcagagga ggcagctttc ctcttttcct aatgggccaa actttatgga tagtgcttgg

19381 agcccaatac cttctgtaag aatgttcaaa agtccttcct tttcctttca ttaaaaaaaa

19441 atccagcctg tatttattat tgagaattta ctgtgtattt gtcagtgtga taaacccttt

19501 ttatgcacta tttaatcctc caacaatctc atgagatagg tactatcaca actaccctca

19561 ttcaactgat gtggaaacta aagcttaggg aagttgagca acttagccag atcacaaaac

19621 cagtgactgg tggagttggg acatgaactg agattttgcc tgggactgga aactgcaaac

19681 gttcatgact tctgtgcctc ctggcagtcc tagaagatgg ttggaggttg tggagaaagc

19741 ttttccacca ttaaggtgct gaggcctgga ggctgtgcca caccttcccc accgcacata

19801 ccaggtttcc agggtttgcc cagccaggag gtcacagtaa caccaggtgg aagaggctgg

19861 tcaatgccct cccagtacac gccaccatca ctggtctcag ccacattggt aaaaatagtg

19921 ttactctgga ttgtagccat ggcgttggga ttggtggtgg cagaggtacc agggg^caacc

19981 ccaaagaagc cgttctcagg gttgatggcc cggagtcgac ctgttaggag aagtgaaaga

20041 aaggaaggac caaacaagga aaacttgaga tgtaagacca aagaaggtcc tgctccaccc

20101 acccctgtcc tgagatggac ccaagatctg atgaggaaag gctgtctctc gggtggctgg

20161 gctgccagag gaatggagca gggttgggtg ttcaggaaac tttctcactg acaaagtcat

20221 tgtgaacact agagaggaga ccccgaggct ctgacaagag ccagaaccaa gagtatgatc

20281 tgagagtccc tcaccttcac tgtcaaacct catccaagca atatcatccc ccacacactc

20341 cactttccag cctggcagtg caggccgcat catagccagg ttggtcttgc cacaggcact

20401 agggaaggcg gctgccacat agcgcttctt ccctgcaggg ctggtgatgc ccaggatctg

20461 tgcattgggg agagagggaa taagaaagag gtcaccaaaa gtcagggcat ggtctgcatg

20521 gggaagggca gctagggtct aatagggagg accaatgttt attaactata taagtgattg

20581 ccctgccatc aggcctgggt tagtggcagg ggaaacctca acattcagca gcctgaggcc

20641 aaagctttgg gccttcaagt agagtccccg cccaccagcc tggcacctgg caggagggag

20701 gtgaggctga ggcagactgg ccaggcccca cccctgccct gtccccggca gctgccctgc

20761 ttctcaccag gccctcacca gcatgtgctc tgccagccag ccctcatccc gggccagccg

20821 agaggcgatg cgtagggcaa agcacttctt gcccagcagg gagttgccac catagccgct

20881 gccgaaggag atgatctccc gctggtcggg cacgtggcca atcagggttt tctctgggtt

20941 gcacggccac tggctcactg gctcccctgg ggacaatggg gtcatggggc tgctggcagc

21001 agcatcagga actggggtgt cggggccaga gggatcaggg attggcagga gagacacttg

21061 ggagcagtag cagcaccaag aggtggctga agccccccac ctcataataa ccatcagctt

21121 ccatgcccct attcttggac acggattttt ctcaacctag tgcaaagtgc tctgcaaaca

21181 cagccttatc cctctgctgg gactgctgct ctggatcttg ggaattaggt tgccagagag 21241 gacaggttct tagggtctgt ggagaggtag gatttgggat ttcctctgag tacaagaagg

21301 cctctgtgtt ccccttgggg cagagcaggt gcttaccttg tcctgtcagg ggctggccca

21361 cggagtgcag acacttgaca aagtcaccat ctcccagggc ctgaagcaca ggtgtcccca

21421 gtcgggtcat aatacgcatg cttgccacca cataggctga gtcagtgagc tgcaccccga

21481 tgcgggacag cggggagccc acaggaccca tgctgaatgg aagcacatac atggtgcggc

21541 ctgtggagga aggcatcccc tggatgaagg gttccaaacc tgttcacatc ttccgtgccc

21601 ttgccactgt gcccctgccc tggttaccct gcatgcagcc tggaaacctc tcatccacag

21661 ctcgctggaa atcagctggg gacatccagt tgcccagctg cccacgggcc ccaccaggcg

21721 ggagtggtac cgtgtcccgc tgagaaggag ttacaatcac cgtcttgctc tctactcgtg

21781 ccacatcctt ggggtctgtg cgggccagcc agctggggga gaggggattg tcaggaagat

21841 ggtggccagc aggtttccct tgtccctaga cccacttttt caagtccaac tttgactcca

21901 ggtcttctgt tggccttatg tttccccacc caatcacatc tcagtccctt cctccaggct

21961 ttgtcccatc taaggggtgt tccccaaggt cccagccctc tgggggtctc tgcccctggc

22021 aaaagctccc tgaactggtg aggaaaaaaa aaaaaagcaa aggatcttat ttatatcagc

22081 ttcttcattt aacattaaaa taaaatacaa actacaaact aggctgggcg cagtggctca

22141 tgcctgtaat cccagcactt tgggaggctg aggtgggtgg atcacttgag gttaggagtt

22201 tgagaccaac ctggccaaca tggtgaaacc ctgtctctac taaaaataca aaaatcagct

22261 gggtgtggtg gcgcatgctt gtaatcccag ctactccgga ggctgaggca ggagaattgc

22321 ttgagacaat tccccggagg cagaggttgt tgtgagctga aatcatgcca ctgcactcca

22381 gcctgggtga tacagtgaga ctccatctca aaaaaaaaaa aaaatcccag tttctttctt

22441 tttttttttt gagacagagt ttcgctctta ttgcccaggc tggagtgcaa tgacgcgatc

22501 ttggctcact gcaacctccg cctcttgggt ttaagtgatt ctcctgcctc accctcccaa

22561 gtagctggga ttacaggcat gcgctgccat gctgggctaa tttttgtatt tttttttttt

22621 tttttcagta gagacggggt ttctccatgt tggtcaggct ggtcccaaac tcccgacacc

22681 tggtgatcca cccgccttgg cctctcaaag tgctgggatt ataggcgtaa gcaaatccca

22741 gtttctgggt ctgtttaccc caaagctctc attcttgttc attccattgg gatgttctag

22801 ttaagttgat tttgccttgg tgaacccaaa gtggcctcag tgcacctatc ctgcaggttg

22861 tggagcccaa ggcttaccag ttattgtact tggggagctt tcggatgagg ccctgctgct

22921 ccagcagggt cagtgtggca gtattctcag cctcagttcc atcacagatg tggatgccct

22981 ctggttggca caggcgggca ctgtgctcta caaaatctcg aatgccagtg ggaagctggc

23041 ccagatctcc actaagcact cgcagggtct ggatgctacg gcatgatggc cagcccaagg

23101 ggctcagccc atgccagtta agcctatagg agaaacagag gcaatgagct agttgctgtc

23161 atcgagagtg gacaaggcag gtggtggaga gacagaaagc ttgggctgca gcctgctaaa

23221 acatgtctgc atgcagggac catgaagagc aggttgcagt tggtgtgtgg gatgtgttca

23281 ggacacatgt atcttgtagc ctttatgagg cttgaggagg gtgtctcctg ctcagcgagg

23341 gacagatatg gccaaatgag caaaaaaggc acatgcctgg ctgtgtggaa gtatacatgt

23401 aggcatgctg atgttactgc ctgtgtagcc catgcccatg tttaactgtt taatcaacgt

23461 ttagaaataa gtagggagat ggctgggcac ggtggctcat gcctgtaatc ccagcacttt

23521 gggaggctga ggcaggtgga tcatgaggtc aggagatcaa gaccatactg ggcaacatgg

23581 tgaaacccca tctactaaaa atataaaaat tagccggatg tggtggcgtg tgcctctagt

23641 cccagctact caggaggctg aggcaggaga atcgcttgaa cccgggaggt ggaggttgca

23701 gtgagccgag atcgggacac tgcactccaa cctgggcgac agagcaagac tctgtctcaa

23761 aaaaaaaaaa aaaaaaaaaa aaaaaagaaa tgagtagggg cctttggccc aagtgtcaag 23821 acaagaagtt gggcccaggt gacaatttca tatgtctggt atttgaagcc tcatccattt

23881 tatttccatt ttcaagtata gttttttata aattaaaatg gcagctgtgt ttattctttc

23941 cttctggtgt gtatgctgta agactatgaa agccccaagc aaaggtcaca gtctttccag

24001 tgccaggttt ctagatgagg gctcttgcac agagaatagg tgactggatg ctgggcttcc

24061 aggccctgat ctgctgctgc ccacttggac caacaatggt tgcataaaat gttgccctcg

24121 ggtaagtatt tctgaactct gcggaaggtt gaccactggg gaatgagtgt cactggctgg

24181 ggttcatggg cttaattctc aggaggtcag cgtcctgttg tctgtcctct catggccctg

24241 ggagacagag aagcaacgtc tgctctgatt ggcctggact caaggtggag taaacccgag

24301 agagcaaaga ccatgaaggg gatgttactc tcctcctcta tcaaggcctc gcccatcaga

24361 aactgccccc acacccagga ccacgtgacc ccgtgcttcc cctggctctt cttcctagag

24421 gccacatgcc tagagagctt tggtgttggt gggagggtgg aacagcagct tgctggcagc

24481 tcagactcag agggtgttag cctggggaag gaagaaagct cagagcatgg atacgggagc

24541 ttgagtctgt ctggggtaga agctggggtg gtttccgcta aagcaggata gatgatcagt

24601 gtggccccag gggcagggga ggagggctac tgggacctgg gtctgttcct gcctcagagg

24661 gaagttctcc gtgagcatca gttttcaagc acacagtctt gaggacagcc tctctctccc

24721 tggagcctgg ctcacgttat ctcttgtgaa gcccccacac cctcttgcag ccctctaagt

24781 gggacgtttc cttcagagac cacccacaag ggctgcccaa gtggaacggg ctgtgcgaca

24841 gatgtgggtc acttccgact ctgggacaaa agctgagaag agaggcctca gagaataaat

24901 aggagactaa gaggacaggc agacaaacag gacgagcctg ggggctccag gcctggacgg

24961 aggaagaagc cagctatgta agaaacgaag gcaacttggc gccctcctcc caccttgctt

25021 gcctccctcc cctgggccca ggtccctcgg gctgctctgg acttcccgcc aggttccagt

25081 ccttccagtc cgggagaggc cgggaatgga tatttaacct tccaccttct ttagggggct

25141 ctgagcttag tctcccgggg ctcacgtacc cttgtcccca gggccctgct gcctctcgaa

25201 gtaccctggg gcgctggctg agagcccgtt agaggtccga gggaggtatc tgggaaggaa

25261 aggaggatat gcggagacct ggctcagcag agaggagcga ggagagagag gaagttctga

25321 gagtggcaga tagagatcgc gcggtcagag gcgagcgagg cagagaaggg gaggtgcagg

25381 cggaaacgag gagcaccggc cggctgccct cccaagagac gcgcggcgcg ggaggctgga

25441 cccacagcag tcgcccctgc ccgcctccgc ctaggatggg aaacctggcg gaggctgaga

25501 gcaggagagc gccactggca gccccgaggg ggcgagcgca gcggaaggtg cgggtgggcc

25561 ccgggccggg ggtcactcac cgcaggccag ggcggtacaa tgcggccatg gcacctgggc

25621 aggggetgcg gggtggccag gaaccgagcg gagccgggga ggtatggaag gcggggaagg

25681 agggagcgcg gcgaagcgag ccacagcaga ggctggcggg aggcgcttaa aaaggagggg

25741 ggcaggcgcg ggggcggaac ctggtagggc tggcactagc tttcctccgc cccgctggca

25801 ggccagctcc ctcggccccg gggctccagg ctccaggtca gcccagccgc cgccccaacc

25861 cctgcctcca aaccgccaaa cccatctctc ccattggagg actgggtcca ctcttcggcc

25921 cccgacttcg aggccccgcc ccccatccgc caccgccctc ccagccgcac atgatgtaac

25981 tttgaggact cttgctcccg ggagagacac taggtggcaa acaagctagg aacgaggggc

26041 gaggccaggg gcgggagact acaggggcca gacgtctaga agttaggggg ttgggcattg

26101 cctttccagt tcctcctcct ggggacgcga caccctgagt cctgggcctt tctgtagtcc

26161 aagtgcccaa tgctaaggtc cacttagtcc ccaactccta aatccccgtt tctcctcctg

26221 atcaaggctc ctttctgcct tgccatgaaa tggtggagtc tctctacccc tgggtctctt

26281 ccttggtcct ctgatctggt cattctcctc ccccggcaca tcccctcttc ccctgacgga

26341 gtcttactct gtcacccagg ctggagtgcg tgcagtggcg cgatctcggc tcactgcaca 26401 ctccgcctcc cgggttcaag caattctcct gtctcaacct gcagagtagc tggaattaca

26461 ggcgcctgcc accacactcg gctgattttt tgtatttttt gtaaagacgg ggtttctcca

26521 tgctggccaa gctggtctcc aactcctgac ctcgtgatcc gcctgccttg gcctcccaaa

26581 gtgctggaat ttacaggcgt gagccaccac gcccggcctg gtcattcttt ctctccacaa

26641 tccaatgagg aaactcatct gcccacccaa ctaccatcat aaagtcctca ggtgatttct

26701 caagctatac ctccattggg acatctctct ccaacctgag accactggat atcacccaca

26761 atgctatata gcacctcaaa ctcagcatat ctatgatggg tgtctgctca gcatccactc

26821 gccttttctg gtaatagcac cctgattttc ttttggggaa ccatctcctc agtctgtatt

26881 gtcatgtggg tctgatgtca cccctcttgg gggtaggctt gcagcttagc caacttttgt

26941 gttcctcact ctggccacag caatcaagga tgttcaagga tgggcatgag atccctctct

27001 gggacttttt ctaaaacttt ggaagttagc tctctttctg ctgagttgct gagctggtag

27061 aatataagcc tggagctgct gagtgttagc ttggcactgc aaagcaggaa cctggctgag

27121 acagaagcca gcctagagga agcagagcag ggagatggcg agcaatggat tcctgatttc

27181 agttacagga attgatgcat ttcttctctt gctcaagcta attttaattg gagttctgcc

27241 acttgcaaca gaaacagtct tgattctcag atttcttgga gttctatgct ggaacatagt

27301 gggataagga gagctggtct ccagccacgg gcttgctact ggtccccgtc tcttccccct

27361 caggctctag cctacactca acctcaggta tatgtatttg gatctctttc cccccagttc

27421 atagatccaa actctgtcca cacttaccca gctgcaaaca catacctctt ggccaacctt

27481 tgggcctaaa ggtgtgcaca ctggtggcag catggttgac ctgaaggtca tcatcttctc

27541 cataatcctg cacctgctct catggtttcc tacctcagca aatggcacca tcatctggcc

27601 agttgcccca aagggaatca gtctagacta ttcttatcct gcatagctaa tcagatgcca

27661 ggacctttgg aattgtactt ctcaattagc cctcaagtct gcaactatcc ccatggccag

27721 ggccttagtt cagggcttat ctctcacctg gcttcaggtg atagcttcta aggcacttct

27781 tggactcctg cctctccaaa caccaacccc tctccctacc aagccactcc ccacactgtg

27841 catgcaaatc cacttggaaa gttaagagag aagaataatg tttatgctat ggacagtagg

27901 tgccaggctc tgtgtcagca actctgcatg cataatctca ttctgtttca caactatatt

27961 ttacatatgt ggacacgggc ttaaagagtc atttgctcgg tcaccctaag ggaagaatca

28021 aggaaaaagg gaggcaggac cccagaagta tgccaacata taacacccta aatcaaaggt

28081 caaatgccgc acttgacctc caagatgcct gcttgggcct cttccaagtg tactttcctt

28141 tctttcattc ctgctctaaa gctttttaat aaacttccat tcctgttctg aaaaaaaatt

28201 attcatgaat attttatact tttatcctat tatgaatgaa atgttctaat tgtttaccac

28261 aagtacatac aaataaaatt gctttgtgta tattgaaaaa aaaaagagtc atttgctcaa

28321 agtcacacaa atggttagca ggagaaatgg aatctgaatc catatctttc tgactctaat

28381 taccaagtcc ccctctccct ataaatccta tactgtcccc ttgatttacc attaaaaatt

28441 agatcatatc actcttaagc ttaaaatctc ttccaggctc tttccatcac atgatatctt

28501 atgtccttca taatctgact cctatttatc taatgttgtc acccccacat ccccacttgc

28561 ctccttttct cctacgtact gaactctgaa gtttccagag cacttctgtg aactttttct

28621 gggcatctgc actcaaatta aacacacacc atcctggcca acatggtgaa accccgtctc

28681 tactaaaaat acaaaaatta gcttggtgtg gtggcgcacg ccggtagtcc cagctactca

28741 ggaggctgag gcaggagaat cgcttgaacc cgggaggcgg aggttgcagt gagccgagat

28801 cgcaccactg cactccagcc tgggcaacac agtgagactc tgtctctaac acacacacac

28861 acacacacac acacacacac acacacacac acacaggcct tttctgttcg tcccttagaa

28921 tgaacatcta tttcatctgt gttgcttcta tcccatgtat atgcctatat ttttgtactt 28981 tactatactg tagttacttt tatatctctg taatcctcac tagagtgatg atttcagtct

29041 tgtttttctc ctagttccca gcagtacctg gcctataaca ggcacttaat aaaagttggt

29101 tgaataaaag aatgaatgag tgccacgttt ctgaattctt cttccccacc attactttct

29161 tctctagctt taggcagatg gagcaagggg ctcacgtgca gatttccttg cactgctcat

29221 ttccccttat ctgccacaca cttttcttta gttccctgcc gggagagcct ggcccaccag

29281 ctggccctat gtccaaggag taccaccagg gggcagagtg tgatctgcat tgtcctctct

29341 ggggcctctg ggttcatccc tgaagtctca cagcccgagt agtgatgctg tggtgtgtag

29401 cccagattcc cgcttcagga tcatgatgct cattgttcca gctgctgagt gttggctgct

29461 gatagctcac agctgagtac ttccttgagc actatctgtg gccaaaggga gctgtctcac

29521 caaatgcaat gcccgcttcc tgggggcagt atgtatccaa tgaatggttg ttataaagta

29581 cagaggccta gccctttgtc tcagtttggg acaactctga aggtccatcc cagccccaga

29641 gctctttagg ctcagctgat tacaactgca gtgccattca actgttccct ctcttgggtc

29701 ctgctactct ttctttgaaa gtcccccccc gtgccccccc cccccccccg agtaaaccac

29761 ctgcacacaa gtttctgtct cagagtgtgc ttccagttgc aaatattcct caggctcaac

29821 ttggttacgg cattagtcct ctaatctgac tcttatttca ttgttgcctt cctccacaaa

29881 cacaaagtgc tgacatacgt gatccttccg aacacaaatc tgatcacatc atgtctctac

29941 tgaaaaccct tcagtagctt ctccctgctc ttagaataaa gcccaaaccc ttaatgtgac

30001 ctcccaggct ctgcatgatc tggcccctgc tgacttctcc agcctcagct atcactgttc

30061 ttcccacctt ccaggctttc tactctctgg ctacgtggat cttttctcag ttttgtgaac

30121 atgtctacat ctttcctgcc tcacaacctt tgcatatgtt gtttctgccg cctggaattc

30181 ccaccctgtc cacacctcca ggcagggtca cttcttattc acctgaccag tcttggttta

30241 gcatgacttt cttaggcaac ctgtccctgt gatgcatccc ctctggcttt gtattagagt

30301 atgtcttcgt ggaagaaaat ttcatagcac cctgcccttc ttcttcttag cacattcatc

30361 tctgtttaaa tggtttgttt ttatgtagac acatctctaa catatgtagg acctgcggca

30421 ggagtacaaa tagaggccct caaaccttgt gtttaaatat ttaaaatggg ctgggtgtgg

30481 tggctcatgc cttgtaatcc cagcactttg ggaggcagag gctggaggat cacttgagcc

30541 caggagttta agaccagcct gaacaacata gtgagacttc ttctctacaa aaaataaaaa

30601 agtagccagg catggtggtg tgcacctgta gtcccaactg cttgggagac taaagtggga

30661 gatagcttga gcccaggagt tcaaggctgc agtgagctat gattgcacca ctgcactcca

30721 gcctgggtaa cagagcgaga ccctgtctca aaaaaaaaag attaaaatgt ataactcagg

30781 gtcggcgcag tggctcatgc ctataacccc agcgctttgg gaggctgagg cgggtggatc

30841 atttgaggtc aggagttcaa gaccagcctg gccaacatga aaccctgtct ctactaaaaa

30901 tacaaaaatt aggccaggcg cagtggctca cgcctgtaat cccagcactt tgggaggccg

30961 aggcgggcgg atcacgaggt caggagatcg agaccatctt ggctaaccca gtgaaacccc

31021 atctctacta aaaatacaaa aaaaaaatta gccaggcatg gtggcgggtg cctgtagttc

31081 cagccggagt ctgaggcagg agaatggcat gaacctggga ggcggagctt gccgtcagcc

31141 aagatcgcgc cactgccctc cagcctgggg gacagagcaa gactctgtga aaaaaaaaaa

31201 aaaaaaaaaa attagccagg catggtggtg catgtctgct acttgatagg ctgagccagg

31261 agaatcactt gaacccagga gacagaggtt gcagtaagcc gagatcacac cactgcactc

31321 cagccagggc gacagagcta tctcaaaaaa aaaaaaaaaa aagtataatt caggctaaca

31381 aactgttaca caaaatatat tctgttcttc taccttgaca aatatattga caaatacata

31441 cttcacaatg gcctggaagg ccaggttcag atttgtattt tatttggaat cctcagggtt

31501 ctgcaccaga atgtgatggt gtagggggat gtagttttgg ccactggccc tcagctgggc 31561 ccactttgcc tcctacccct gacaccatct tggactgcaa ggggccttgt gtggaaccat

31621 gtggacctcc catcaggcca tcccaagtct gtccacagtc tcctgcaagc agctacccct

31681 tgtctaccct ttgacctcag aatgtgtgaa ccagtcatgt ggcccagccg tgggagggca

31741 aacccaggga agaggttcac gtgggcccta ggagtgggct tgggcctttt ggacagggaa

31801 tgcttagcta tctggagtgt ggtcctgaga ggattaaggt atgggcagag gatagttgga

31861 ctcctcaccc atgtggtggg acatagccag aggaggccag agtggagcct tctaaagcac

31921 agggcccagg gcaggggcca ctcctgcctg ggcctaagga caataattta tttattattt

31981 tttttttgag acagggtttc tttctgtctc caaagctaga gtgcagtggc accatcatgg

32041 cccactgcac cctcatcctc cccaggctcc tgatcctccc acctcagttt ttgtattttt

32101 agtagagaca gggttttgcc atgttgccca ggctggtgtc gaacttctgg gctcaagcga

32161 tccacccatc ttggccttcc aaagtgctag gattacaggc atgagcctct gcacccagcc

32221 aaagggcaat aatttgatgt ttctctgttt catcagtctg taagtttctt gaagtcaggg

32281 ctggtattag tgtaagttac tgaggggtcg ctaatgcctt actcaggatg tggtgcagaa

32341 taggtgttta atatgcattt ttcaaaagaa tgtaggaggt acaccaagaa gaggttgagg

32401 atttggatct attactgaag atctagatac catgagccag aagtaggaat gagaaccagt

32461 ctttcagggt tccaaaaatg agagtgcagg acagaggaga ggaggtagca tcctggaatg

32521 ctgactgtct cattttctct ccttcctctt cattttctac cccactcctg caggcccttt

32581 gcctgcctgc ccctctttcc ccatggtcca atatcactcc atccatatct tgctaccttt

32641 ctgtcctttg gccactgctc ttcctctgtg gcctatgtgt ctatttcttt gtattggtgt

32701 ctaggtcttg gtccccaatc ttatgtcctg tcccttcctt ctaccactct tactgttcta

32761 gtccttgttg tcccctactc ccttcttggt cctctcctgg aagacttcct ttctttatcc

32821 atctcgtcca tgtgcctaca tctttctgtc tcctttaatc caaatcccca tggagaccct

32881 gacttgtcct gtcctctatc tcacatctac acctactgcc tccttagtct ccttcctctg

32941 ttctattatt cctggagtca cactggcttc atctttttca tccctagatg tccttgagcc

33001 cattagtctc agtattcatt tctcagtctc cagtggatat gggagatggg ctctgctttc

33061 tgggtcccca ctgactgcca cagactctcc tctctgtgcc cttggtcctt tcctctggcc

33121 cagactgggc ccaaccctca ctcccccagc tcctttccct ggtttagtgg tgcctggtct

33181 gagccccaag cagctcaact cgcctgagct tgtttagtct gactccctta gcccgttagc

33241 agcttagccc tcccacctct cctttgagcc ctccagtaaa caagaggatc agtgcagacc

33301 tgacagctgc tggttggggg caagtgtctg gccaggccag agcaggcatg aaaaaggggc

33361 tgcactcaac acttcccatt ccttcttcaa ccagggaaat tgtagagtct ctttcctggg

33421 tgtccacttt ggagagctac attccatggg catgtgtgtg gggtcctgcc tctccatgat

33481 cctcattccc attttgtggt tggttcctag gccttaggct tttttattct tttgagaaaa

33541 cagcagtaga tccaggcagt cacacagaga agcttgaatg gtgacgatgg tttctgctcc

33601 ttaaaacaga aacccaggtg tgggaagaga gtgatgtcat ctgatctagg ttttccaata

33661 tgtgtatgga aagcatggta ggagagacca catacacatg cacagacaga catatataca

33721 tgcctcccca gggaagctgg gtagactgca ggcattctgt gcagttctca aactttcttt

33781 cttttctctt cttttttttt tgagatggag tctctctctg ttgcccaagc tggagtgcag

33841 gggcacgatc tcggctcact gcaacctccg cctcccaggt tcaagtgatt ctcctgcctc

33901 agtaggagta gctggcatta caggtgcctg ccactatgcc cagctaattt ttgtattttt

33961 agtagagatg gggtttcacc atgttggcca ggctggtctt gaactcctga cctcaagtga

34021 tccgcccacc ttggcctccc aaaatgctgg gattacaggc gtgcctggcc tttaaacttt

34081 caagcatgtc agacttgctg gaagggcctt aaaacacaga tttctgggcc ccacccccgg 34141 agtttctgac tcagtagatt taaggtgaga tcaggtaaca tgtatttcta gcaagttccc

34201 aggtgatatg gatgctgctg ctctggggac cacacttcag aaaccacctt cttagataat

34261 ctgtatctgc cagcttttcc ccaacatata aggctgggac caggttaagt tttataagtg

34321 atgacctttg gggtctgatg gacacccagg cctctgtgtt gagaaagagg aactattaac

34381 cccatgggag tatttgggtg gcctcagaag gtaggaagtg gcctttaact ccatagaccc

34441 tatttaaaca gcttcggaca ggtttaaaca tctccttgga taattcctag tatccctgtt

34501 cccactccta ctcagggatg atagctctaa gaggtgttag gggattaggc tgaaaatgta

34561 ggtcacccct cagccatctg ggaactagaa tgagtgagag aggagagagg ggcagagaca

34621 cacacaggga aaaggtgaga gaaagcagag ggcatatgag gccagttaca aaagtgtctc

34681 cgacggactg gtggtgatta gatttcaatg tcccttgagg cccagaagga aggaacagag

34741 ttccctgttc tcttttttcc tactctttct cccaagctgg attagcaaga atgggagaga

34801 caaaatatgg tggtcaagtc ctggagatcc cctgtgggaa aagttgcttt cctttctgat

34861 gcgacggcta tgaggggcat aagaccagtc agcatgaaga tggggcagag gtagaaatgt

34921 gaggattctt gaaaatagga aacagatcca aaaggcccgg tgagagtggg cgtgggagag

34981 cttatagtct ccaaagacag atgaggggct gcagggcctt ggaaggtggg ggtgctggga

35041 gtttgtctgg tcctcttttt ccaatcccag gctatgcatg gctagggctg agggttgtgt

35101 tcacatagag gggtggtggt tcagacccat gtttctgtcc gaactgggaa actgagcact

35161 tgggagtcgg atcatcagtg aggagctttg ttggggaaga tgctcccttt ccttctttga

35221 gaataagcac acttgtactc tcctctctct ccatttcccc caaccccagg ccaataagat

35281 atgcaaatga tctccttacc ttattggttg gaggcctcag gaccacagat gacctcagag

35341 agctggccct ttaagaatgc cctttgggct ctgtgcccac agccccctgg agctgagcag

35401 aggcaccagg ccctgctcca tggagccttc agtctcctgg gaagctgtgc ctgtctggct

35461 ctggcactga ccacatcctc tcggccattt ctgaagtgag tatatccagg gcctacctgg

35521 gtgtcaggcc gctcacctag aaccccctct ttaacacctg ctctctgttg tctgttttca

35581 agaatcctaa caccttggta ccctactcca ctcccaccct gactggcctg tgtccctcac

35641 tgctatcacg tcagaaagat aaacaacctt ccccactact ccttctgtac atccagttct

35701 ttctggatac ctctctcagt gtctcaccat ctcctttctg ccaagaccca gttcctttaa

35761 tctgtgtctg tttccatccc ctctgtcctc tcaagggtga caggttccat agagagggca

35821 gcaaccctgc cctggaacag gaagagagag gcctgggtct tcctctttgc ctctctgggt

35881 agagcctggg ggtctgacat gtggttggaa agaaggctac ctaggcttgg ggcatctaga

35941 aggagccgaa gtctaagccc tggggatgaa cactccatgc cgcgcacctt ctgggggtgg

36001 ggtgggagtt actggaattg ggagctggac tagagcttgg acttcaaggc tgggaagggt

36061 atctgaggtc acccagccag aagttcctca ggtgaacatg tgccccatga ttgataacag

36121 acaggagagt tccttggtga cataagttta ggacactttt tgttgaactt gaacagacgg

36181 tttactaaag gattgctcag gttaatatgc tcttgtgcta tgtgcatctg taagaggtag

36241 ggataggagg caacatttct caaagttatc tgagcacaga cctcttcata ggtattgttc

36301 agaatcagtg ttctagagaa cactctttgg tatatgttac aaagtctaac tgccctcatt

36361 gaacagaagg gaaaacgggt cagaagggtg aaggtgatct gccaagttca cacaacgtaa

36421 caagttctga atagaaggaa aatcaatctt ggtctgttcc ccttggagag actggccttg

36481 aggaaagatg gtggccagtt gattctgatc tttctagaac taattctagg acctttcttt

36541 tttccatata aagcctcttg cccctacaaa agggattcat tgattgatta atccatagaa

36601 caaatgtaca ctgagggtct tctataggca aggcacttcc ctgagtgctt tgagagacag

36661 agagctataa gaacacatta tccttggctc ttaaaattca cagcatacat gccttcccac 36721 tCftcctclclcLcL tatacactca ctttgtcaca gttaattaag caatttaaaa aaatggcttg

36781 gaaatggaat gatgcctctt gagatgacag acctctcggc atgtctcagc actagggttg

36841 ggagcactgc catactgctc ttgcttttca tagatcctga ggcatcagtg gggcgagagg

36901 ctgtgctgtc ctcttcctcc ttcaggaatt cagctgcttg tcccctgtca ggagcccctg

36961 ccctctgaaa ggttactctt cagcctggtg gggactctgc agtgaacaga gctgcaccat

37021 ccctctggct ttcccaaact cttgctccag ggcacttggg ctttgaggga agagggactt

37081 ggtgaagagg ggatggcagg tggcctccat gtgctccaga cctctcctcc tctttgcagg

37141 tgcactcctc ccagcccagc tccagaatgg ccctgccccc cagccccctg gccatggaat

37201 atgtcaatga ctttgacttg atgaagtttg aggtaaagcg ggaaccctct gagggccgac

37261 ctggcccccc tacagcctca ctgggctcca caccttacag ctcagtgcct ccttcaccca

37321 ccttcagtga accaggcatg gtgggggcaa ccgagggcac ccggccaggc ctggaggagc

37381 tgtactggct ggctaccctg cagcagcagc tgggggctgg ggaggcattg gggctgagtc

37441 ctgaagaggc catggagctg ctgcagggtc agggcccagt ccctgttgat gggccccatg

37501 gctactaccc agggagccca gaggagacag gagcccagca cgtccaggtg agtggtcagc

37561 aagctggcct gaggggaggc agggcaagga aggaggactg cccaagagag gaaggggagc

37621 tcccagaggg ggttatggac tgggacaggg gacagagggc aggagaaggg gagaaggtcc

37681 cttgaaagca atcagatcga gaaaactaca ttctgcttct cccccttttc ttaaaatgga

37741 gagaaatgag taggctgaac caggaggagc agggagaaga taagattagc agagaatcca

37801 aagggaagaa ttgagctggg gagtgggcga ctccgggggg taaacagata catggtgtgt

37861 ggaaaccagg gaaagggtta tgtgtggagt ggagctgggt taagactggt ttagattggc

37921 cttttcaaga cttcgtgctt cccagccccc aaccttctca ggaaggattg ggactcatcc

37981 tattaattac agacacagat gggggtgttg cgggcattag gatgtaatcc aaatcctgtg

38041 agggtcaccg ggttgcaggc ttcaggacag ggaaacgcag cgcgttggag ggttgggggc

38101 tgtctgggtg cgggtccact ggacacaccc gggcctggag ctggatgccg gggatcccag

38161 agacgagccc ggggtttagg tgcgcgacgg gctcgcctga ccgtggccgg ccctgcaccg

38221 tggggcgccc gcctgactgg agcaacggtc agctgggggg cccggggagc gtcggggcct

38281 ggggcgggct ctggaccgaa acagactgcg tggaagggcg agccttccgg tgaaggtggg

38341 agccggggcg gggctgtccc ggggcggagc caggtagcgt cgggccctca gggcagagcc

38401 gggtgcgacc tggcgctgac ccggtttctg cattctccct ccgcagctgg cagagcggtt

38461 ttccgacgcg gcgctggtct cgatgtctgt gcgggagcta aaccggcagc tgcggggctg

38521 cgggcgcgac gaggcgctgc ggctgaagca gaggcgccgc acgctgaaga accgcggcta

38581 cgcgcaggcc tgtcgctcca agcggctgca gcagcggcgc gggctggagg ccgagcgcgc

38641 ccgcctggcc gcccagctgg acgcgctgcg ggccgaggtg gcccgcctgg cccgggagcg

38701 cgatctctac aaggctcgct gtgaccggct aacctcgagc ggccccgggt ccggggaccc

38761 ctcccacctc ttcctctgag ccgttcagag caccttgtgg tgtagtgggg gctgggtggg

38821 gtggctccgc ccaggaggcg gctgcacggt tctctgcatc gttaccagag cgccttctgg

38881 tcctagccac gccctgtatg accgcgcaaa tatccccaaa gcttttgggt cctcaagtca

38941 tgcccgaatt tagatgctgg tcattttctg gagaggggtc ccctcccctt acgaacacag

39001 aaacccagcc cacatgacta gcacgctgag ctctgcaggg accagtgcca ggcactgggg

39061 ggtggaagtg tggtgacaca gtgaatggga ggtggaggag ggttgcagct cccacctcag

39121 tttagttttt aattcagggt tttcaacctg taacacatta aagctgtaat tagcaatgag

39181 gctgtatttt cattctgaag cttgtaacct ccccatttta gcactacaga attttcaaga

39241 tttcaatatc caacaactag atagattagg acctctatcc gagatgcttt ttccctgccc 39301 aaccctgtgg ccttcagggc tcagagcagc aaaggcctga agagtgagct ctgggggttg

39361 ttggtgtggg ttgggagaga gctgtgtgca gaagtctgga aacctgggtc ctagtcccag

39421 ctcttccatg ggatccccct gtcaccctga gcaaatcagt tgcttcctgg acttgtgtta

39481 cttcatctaa ttctcatgtg gattggacga cttctgctcc ctttccagtt ctggcatctc

39541 cccagtatgg aagtcccggt ggtctcccca agaagtcccc aagacaatct cgccaaaggc

39601 acctcctatc ctgctgcagt ttcccagctg cagcctaggc aggggatgca cagcccaggc

39661 gaggaagcct ggcttctctg tgagcacata cgtgggtcct cggcagctcc ctccaggctg

39721 tctgggcctc cagacctgca cagggtgctc ctgccacctc ccacctctct gagggctgag

39781 gtgagacttc tcctgggatg acaatttgct gagagagtgc agcttttgtg aattaaactt

39841 gaagtccagg cagaattcta atgcaataag ctaaatgttc ttgcaattta agaagtgttc

39901 attctttatc cctgcttcag gctactgttt gtgtgtctgt gtgtgggaga gggagagaga

39961 gggagagaga gagagagcaa gaaaaaggga agtccccacc tggagctggg tggcacatct

40021 gttatggaga gccaatggaa atggggtgga ggggcaggca ttggaacaga tggatcctcc

40081 ccctctcacc accaccacca ccaaactcag aagcctcagt ggcccagctc ggcctgtcag

40141 tggctatcca ggtctcacag tttccatggt aacccagtcc tccagttcag caggctaagg

40201 ggagggcaga tggggcctct gggcagagca caggcgttgt gctgggagca gccgcctcac

40261 gagttactgt catctgtcct ctaccggaca taataaatgg gagaagcttt cgggcatggg

40321 ggaagaggct cccatatgtc cctcttcctt tctaccccct ctctccaccc cctccccagc

40381 tgtctcttct cttcccatca cagttcttgc tccctctttc ccctcctcta tcccagctca

40441 tccctcctcc gcttaggagc ctgcaatggg agtcctctcc tccatcgctc tcagaatttc

40501 tctcgtctcc tctcccttcc ccaccattgg ccactggcca tcctctctgg ctctggattc

40561 tctctctgag gggctcacag aacctttagc ttctctttct tcatcccctt ccacactcat

40621 tgttgaaggg agaggatggg gcagggaggg tccttccggg agcccagctc tttttctctc

40681 actctaaatt tcacctgtgt ctatccttgc ctgccctgga gctgtaattc agacccacta

40741 cttttcttgt tcacttttct cacctgcatg gcacacaaag acagacagaa ccatccatag

40801 acacctgagc acacaactag gccacatata cagtaccata cctgggtctc aaaatgatac

40861 tcctgggtta cctgagtata cttaaaaaaa caaagttgca cactgtggtg attaagagta

40921 gtgctttagg tggggcgcag tggttcctgc ctgtaatccc aacactttgg gaggccaagg

40981 aggaagtttg agaccagcct gggcaacata gcgagaaccc tgtcactaca aaaaaataaa

41041 ataaaataaa ttcagatgta atggcatgca cctggagtcc cacctaccca ggaggctaag

41101 gtggaaggat aacttgagcc caggagttcg gggatgcagt gagccataat cgtgccactg

41161 cattccagcc tgggtggcag agtgagaccc tgtttcaagt tgcagttcta ccagtttttt

41221 gaccatgggt gggttaaaat caagcctcac ttgattgtca gaaaaatggt gaaaataatg

41281 gtaaccacct actgagaata aatgcattta aataagcact tagcataaag ttgacacatg

41341 ctacgtaatg atatttgctg ttgttgctga tgtgattgtt aagtgtgtga atctgtgtgt

41401 tcatgcactg cacatccctg agtccatgag tgtgacccat taaggccatg aacagacaca

41461 cacattgaca cacccgggaa accagactgt ataggcctta gatgctccac tatgttccca

41521 attcactagc agacctgctg ccctccttca gtatccccca gcacctgctc ttggcccgcc

41581 ccacagctgt gtgggggctg gggttcagct cctgggggac tcagcagggc ctctctggag

41641 acagctataa gaaagcttca gcctgccctc ctgacagccc gtaattatcc tcctatctcc

41701 tgtaattatt ctccatggtg cctagacccc ctggggttgg gaggtgaggg agggggetcc

41761 tcagatccca ctggcacata gctggggtaa gactgcagag acagcagcct gcctggggag

41821 ctctgtctca gattttgaaa ggtgctggtc ctcatttctt atggtgtgtg tgggggtgct 41881 gcaggctgcc cagtgggcat ctgaggcatg atggttagag gctgaggtgc taggagcagt

41941 tctctttatt aatggctaga aagtcaggat cacccaagga agtcactgag gggccacagc

42001 attgaagggt atggggtttg gagagatagg agcaggaccc accactcacg tccagaaccc

42061 agggggeaea cctggtccaa gaggtggagg cattggtcac tggagtcacg agggtcagga

42121 caggcacctg ggagggggtg ggagagtcat aagcactctc ttctacacta cacccagcct

42181 agttgggctc taccaagccc cactttgcac cccctgtgtc cactcctttg gaagccccag

42241 gactcactga gaggctgagg gagtgtcggt ccggagggag gcagtcacgg gctagggctg

42301 ggagtcgtag ccagtgtgca gggcctggga gccccagggc tgatgccctg gctggcgtag

42361 tactccacca cctgccgtgg cacctcagcc aggacacact tggcgagtgc agagggggca

42421 gcctagggtg gggacagtga tgtggttagg ggtgagagga agagtagggg aggcagggtc

42481 atctgaggga gaagtggggc ttcagggtgg ccagctgggc tcccgcgtcc ttagcaggac

42541 atcaaatggt ggaagacgaa ggtcagattt ggggggatga gaagctgagg agtccttcag

42601 ccggaagggg cccgggggac tcacatcctt gaagtctcgg aagggcacga actggacaat

42661 gtctcgggct gcaggcaccc ctcgggggca gcgcaagggg ccgtcgtcgc catccagcag

42721 ccgcatgtca gagaagtcag cattgcccac gcctacgatg atgatggaca tgggcaggcg

42781 ggaggcacgc acgatagcag tgcgagtctc agccatgtcg ctcaccacac cgtcagtgag

42841 caccagcagc accgagtact tctgagggca agcagagagg gtggaagggt tggctttgtg

42901 tgcaagggcg ttgacaccgt ttccaccaag gtgcagaccc tggccctggc accacttaaa

42961 gacacatggg acagctcctg tttgcccgcc atgtcttcct accgtggctt ggccggtgct

43021 ctgctcccgc tgggccggct cagccacacg gttgatgatg ggggccacat tggtggggcc

43081 gtagagctgg atctggggca ggcaacgacg gtaggaggcg atgacccctg agatctctgt

43141 gagtgaagag aagttgtgaa gctggcagaa atgagcaggg ccccctcctg acactcctta

43201 aagcaaggaa atgcttccag gtctgaggac tgccctcccc tcgagttcct tcattaagca

43261 gtgggagagt tggatggtcc cttggcaggg agagttgggt gcagatgctg ggcctcggcc

43321 ctggaagcc

SEQ ID NO: 2 has the following nucleobase sequence (5’-3’)

1 cgccgggggt gtgtacgcca tgagggggag gaagccgaga agcacctccc actgaagccg 61 gcggtaaccg ccccacgcga gaccacccac cagccaggag cggtcgcagg acccgcagcg 121 gcgcgaggcc ttgtttacac cgactgccgc cactgcgttg gccaaccgct cctccgtcac 181 gcgcttcgct cgtcacgcat ctcgcggctt cctggccggc gggctgcaaa cgtcacacga 241 cggccccgcc cccttggcca gccaactgga cagcggagca cgatgattga taggccaata 301 agatatgcaa atgatctcct taccttattg gttggaggcc tcaggaccac agatgacctc 361 agagagctgg ccctttaaga atgccctttg ggctctgtgc ccacagcccc ctggagctga 421 gcagaggcac caggccctgc tccatggagc cttcagtctc ctgggaagct gtgcctgtct 481 ggctctggca ctgaccacat cctctcggcc atttctgaag tgcactcctc ccagcccagc 541 tccagaatgg ccctgccccc cagccccctg gccatggaat atgtcaatga ctttgacttg 601 atgaagtttg aggtaaagcg ggaaccctct gagggccgac ctggcccccc tacagcctca 661 ctgggctcca caccttacag ctcagtgcct ccttcaccca ccttcagtga accaggcatg 721 gtgggggcaa ccgagggcac ccggccaggc ctggaggagc tgtactggct ggctaccctg 781 cagcagcagc tgggggctgg ggaggcattg gggctgagtc ctgaagaggc catggagctg 841 ctgcagggtc agggcccagt ccctgttgat gggccccatg gctactaccc agggagccca 901 gaggagacag gagcccagca cgtccagctg gcagagcggt tttccgacgc ggcgctggtc 961 tcgatgtctg tgcgggagct aaaccggcag ctgcggggct gcgggcgcga cgaggcgctg

1021 cggctgaagc agaggcgccg cacgctgaag aaccgcggct acgcgcaggc ctgtcgctcc

1081 aagcggctgc agcagcggcg cgggctggag gccgagcgcg cccgcctggc cgcccagctg

1141 gacgcgctgc gggccgaggt ggcccgcctg gcccgggagc gcgatctcta caaggctcgc

1201 tgtgaccggc taacctcgag cggccccggg tccggggacc cctcccacct cttcctctga

1261 gccgttcaga gcaccttgtg gtgtagtggg ggctgggtgg ggtggctccg cccaggaggc

1321 ggctgcacgg ttctctgcat cgttaccaga gcgccttctg gtcctagcca cgccctgtat

1381 gaccgcgcaa atatccccaa agcttttggg tcctcaagtc atgcccgaat ttagatgctg

1441 gtcattttct ggagaggggt cccctcccct tacgaacaca gaaacccagc ccacatgact

1501 agcacgctga gctctgcagg gaccagtgcc aggcactggg gggtggaagt gtggtgacac

1561 agtgaatggg aggtggagga gggttgcagc tcccacctca gtttagtttt taattcaggg

1621 ttttcaacct gtaacacatt aaagctgtaa ttagcaatga ggctgtattt tcattctgaa

1681 gcttgtaacc tccccatttt agcactacag aattttcaag atttcaatat ccaacaacta

1741 gatagattag gacctctatc cgagatgctt tttccctgcc caaccctgtg gccttcaggg

1801 ctcagagcag caaaggcctg aagagtgagc tctgggggtt gttggtgtgg gttgggagag

1861 agctgtgtgc agaagtctgg aaacctgggt cctagtccca gctcttccat gggatccccc

1921 tgtcaccctg agcaaatcag ttgcttcctg gacttgtgtt acttcatcta attctcatgt

1981 ggattggacg acttctgctc cctttccagt tctggcatct ccccagtatg gaagtcccgg

2041 tggtctcccc aagaagtccc caagacaatc tcgccaaagg cacctcctat cctgctgcag

2101 tttcccagct gcagcctagg caggggatgc acagcccagg cgaggaagcc tggcttctct

2161 gtgagcacat acgtgggtcc tcggcagctc cctccaggct gtctgggcct ccagacctgc

2221 acagggtgct cctgccacct cccacctctc tgagggctga ggtgagactt ctcctgggat

2281 gacaatttgc tgagagagtg cagcttttgt gaattaaact tgaagtccag gcagaattct

2341 aatgcaataa gctaaatgtt cttgcaattt aagaagtgtt cattctttat ccctgcttca

2401 ggctactgtt tgtgtgtctg tgtgtgggag agggagagag agggagagag agagagagca

2461 agaaaaaggg aagtccccac ctggagctgg gtggcacatc tgttatggag agccaatgga

2521 aatggggtgg aggggeagge attggaacag atggatcctc cccctctcac caccaccacc

2581 accaaactca gaagcctcag tggcccagct cggcctgtca gtggctatcc aggtctcaca

2641 gtttccatgg taacccagtc ctccagttca gcaggctaag gggagggcag atggggcctc

2701 tgggcagagc acaggcgttg tgctgggagc agccgcctca cgagttactg tcatctgtcc

2761 tctaccggac ataataaatg ggagaagctt tcgggcatgg gggaagaggc tcccatatgt

2821 ccctcttcct ttctaccccc tctctccacc ccctccccag ctgtctcttc tcttcccatc

2881 acagttcttg ctccctcttt cccctcctct atcccagctc atccctcctc cgcttaggag

2941 cctgcaatgg gagtcctctc ctccatcgct ctcagaattt ctctcgtctc ctctcccttc

3001 cccaccattg gccactggcc atcctctctg gctctggatt ctctctctga ggggctcaca

3061 gaacctttag cttctctttc ttcatcccct tccacactca ttgttgaagg gagaggatgg

3121 ggcagggagg gtccttccgg gagcccagct ctttttctct cactctaaat ttcacctgtg

3181 tctatccttg cctgccctgg agctgtaatt cagacccact acttttcttg ttcacttttc

3241 tcacctgcat ggcacacaaa gacagacaga accatccata gacacctgag cacacaacta

3301 ggccacatat acagtaccat acctgggtct caaaatgata ctcctgggtt acctgagtat

3361 acttaaaaaa acaaagttgc acactgtggt gattaagagt agtgctttag gtggggcgca

3421 gtggttcctg cctgtaatcc caacactttg ggaggccaag gaggaagttt gagaccagcc

3481 tgggcaacat agcgagaacc ctgtcactac aaaaaaataa aataaaataa attcagatgt 3541 aatggcatgc acctggagtc ccacctaccc aggaggctaa ggtggaagga taacttgagc 3601 ccaggagttc ggggatgcag tgagccataa tcgtgccact gcattccagc ctgggtggca 3661 gagtgagacc ctgtttcaag ttgcagttct accagttttt tgaccatggg tgggttaaaa 3721 tcaagcctca cttgattgtc agaaaaatgg tgaaaataat ggtaaccacc tactgagaat 3781 aaatgcattt aaataagcac ttagcataaa

SEQ ID NO: 3 has the following nucleobase sequence (5’-3’)

1 cgccgggggt gtgtacgcca tgagggggag gaagccgaga agcacctccc actgaagccg

61 gcggtaaccg ccccacgcga gaccacccac cagccaggag cggtcgcagg acccgcagcg

121 gcgcgaggcc ttgtttacac cgactgccgc cactgcgttg gccaaccgct cctccgtcac

181 gcgcttcgct cgtcacgcat ctcgcggctt cctggccggc gggctgcaaa cgtcacacga

241 cggccccgcc cccttggcca gccaactgga cagcggagca cgatgattga taggtgcact

301 cctcccagcc cagctccaga atggccctgc cccccagccc cctggccatg gaatatgtca

361 atgactttga cttgatgaag tttgaggtaa agcgggaacc ctctgagggc cgacctggcc

421 cccctacagc ctcactgggc tccacacctt acagctcagt gcctccttca cccaccttca

481 gtgaaccagg catggtgggg gcaaccgagg gcacccggcc aggcctggag gagctgtact

541 ggctggctac cctgcagcag cagctggggg ctggggaggc attggggctg agtcctgaag

601 aggccatgga gctgctgcag ggtcagggcc cagtccctgt tgatgggccc catggctact

661 acccagggag cccagaggag acaggagccc agcacgtcca gctggcagag cggttttccg

721 acgcggcgct ggtctcgatg tctgtgcggg agctaaaccg gcagctgcgg ggctgcgggc

781 gcgacgaggc gctgcggctg aagcagaggc gccgcacgct gaagaaccgc ggctacgcgc

841 aggcctgtcg ctccaagcgg ctgcagcagc ggcgcgggct ggaggccgag cgcgcccgcc

901 tggccgccca gctggacgcg ctgcgggccg aggtggcccg cctggcccgg gagcgcgatc

961 tctacaaggc tcgctgtgac cggctaacct cgagcggccc cgggtccggg gacccctccc

1021 acctcttcct ctgagccgtt cagagcacct tgtggtgtag tgggggctgg gtggggtggc

1081 tccgcccagg aggcggctgc acggttctct gcatcgttac cagagcgcct tctggtccta

1141 gccacgccct gtatgaccgc gcaaatatcc ccaaagcttt tgggtcctca agtcatgccc

1201 gaatttagat gctggtcatt ttctggagag gggtcccctc cccttacgaa cacagaaacc

1261 cagcccacat gactagcacg ctgagctctg cagggaccag tgccaggcac tggggggtgg

1321 aagtgtggtg acacagtgaa tgggaggtgg aggagggttg cagctcccac ctcagtttag

1381 tttttaattc agggttttca acctgtaaca cattaaagct gtaattagca atgaggctgt

1441 attttcattc tgaagcttgt aacctcccca ttttagcact acagaatttt caagatttca

1501 atatccaaca actagataga ttaggacctc tatccgagat gctttttccc tgcccaaccc

1561 tgtggccttc agggctcaga gcagcaaagg cctgaagagt gagctctggg ggttgttggt

1621 gtgggttggg agagagctgt gtgcagaagt ctggaaacct gggtcctagt cccagctctt

1681 ccatgggatc cccctgtcac cctgagcaaa tcagttgctt cctggacttg tgttacttca

1741 tctaattctc atgtggattg gacgacttct gctccctttc cagttctggc atctccccag

1801 tatggaagtc ccggtggtct ccccaagaag tccccaagac aatctcgcca aaggcacctc

1861 ctatcctgct gcagtttccc agctgcagcc taggcagggg atgcacagcc caggcgagga

1921 agcctggctt ctctgtgagc acatacgtgg gtcctcggca gctccctcca ggctgtctgg

1981 gcctccagac ctgcacaggg tgctcctgcc acctcccacc tctctgaggg ctgaggtgag

2041 acttctcctg ggatgacaat ttgctgagag agtgcagctt ttgtgaatta aacttgaagt

2101 ccaggcagaa ttctaatgca ataagctaaa tgttcttgca atttaagaag tgttcattct 2161 ttatccctgc ttcaggctac tgtttgtgtg tctgtgtgtg ggagagggag agagagggag

2221 agagagagag agcaagaaaa agggaagtcc ccacctggag ctgggtggca catctgttat

2281 ggagagccaa tggaaatggg gtggaggggc aggcattgga acagatggat cctccccctc

2341 tcaccaccac caccaccaaa ctcagaagcc tcagtggccc agctcggcct gtcagtggct

2401 atccaggtct cacagtttcc atggtaaccc agtcctccag ttcagcaggc taaggggagg

2461 gcagatgggg cctctgggca gagcacaggc gttgtgctgg gagcagccgc ctcacgagtt

2521 actgtcatct gtcctctacc ggacataata aatgggagaa gctttcgggc atgggggaag

2581 aggctcccat atgtccctct tcctttctac cccctctctc caccccctcc ccagctgtct

2641 cttctcttcc catcacagtt cttgctccct ctttcccctc ctctatccca gctcatccct

2701 cctccgctta ggagcctgca atgggagtcc tctcctccat cgctctcaga atttctctcg

2761 tctcctctcc cttccccacc attggccact ggccatcctc tctggctctg gattctctct

2821 ctgaggggct cacagaacct ttagcttctc tttcttcatc cccttccaca ctcattgttg

2881 aagggagagg atggggcagg gagggtcctt ccgggagccc agctcttttt ctctcactct

2941 aaatttcacc tgtgtctatc cttgcctgcc ctggagctgt aattcagacc cactactttt

3001 cttgttcact tttctcacct gcatggcaca caaagacaga cagaaccatc catagacacc

3061 tgagcacaca actaggccac atatacagta ccatacctgg gtctcaaaat gatactcctg

3121 ggttacctga gtatacttaa aaaaacaaag ttgcacactg tggtgattaa gagtagtgct

3181 ttaggtgggg cgcagtggtt cctgcctgta atcccaacac tttgggaggc caaggaggaa

3241 gtttgagacc agcctgggca acatagcgag aaccctgtca ctacaaaaaa ataaaataaa

3301 ataaattcag atgtaatggc atgcacctgg agtcccacct acccaggagg ctaaggtgga

3361 aggataactt gagcccagga gttcggggat gcagtgagcc ataatcgtgc cactgcattc

3421 cagcctgggt ggcagagtga gaccctgttt caagttgcag ttctaccagt tttttgacca

3481 tgggtgggtt aaaatcaagc ctcacttgat tgtcagaaaa atggtgaaaa taatggtaac

3541 cacctactga gaataaatgc atttaaataa gcacttagca taaa

SEQ ID NO: 4 has the following nucleobase sequence (5’-3’)

1 gcctcagaga ataaatagga gactaagagg acaggcagac aaacaggacg agcctggggg 61 ctccaggcct ggacggagga agaagccagc tatacggggt ttctccatgc tggccaagct 121 ggtctccaac tcctgacctc gtgatccgcc tgccttggcc tcccaaagtg ctggaattta 181 caggcgtgag ccaccacgcc cggcctggtc attctttctc tccacaatcc aatgaggaaa 241 ctcatctgcc cacccaacta ccatcataaa gtcctcaggt gatttctcaa gctatacctc 301 cattgggaca tctctctcca acctgagacc actggatatc acccacaatg ctatatagca 361 cctcaaactc agcatatcta tgatgggtgt ctgctcagca tccactcgcc ttttctggtg 421 cactcctccc agcccagctc cagaatggcc ctgcccccca gccccctggc catggaatat 481 gtcaatgact ttgacttgat gaagtttgag gtaaagcggg aaccctctga gggccgacct 541 ggccccccta cagcctcact gggctccaca ccttacagct cagtgcctcc ttcacccacc 601 ttcagtgaac caggcatggt gggggcaacc gagggcaccc ggccaggcct ggaggagctg 661 tactggctgg ctaccctgca gcagcagctg ggggctgggg aggcattggg gctgagtcct 721 gaagaggcca tggagctgct gcagggtcag ggcccagtcc ctgttgatgg gccccatggc 781 tactacccag ggagcccaga ggagacagga gcccagcacg tccagctggc agagcggttt 841 tccgacgcgg cgctggtctc gatgtctgtg cgggagctaa accggcagct gcggggctgc 901 gggcgcgacg aggcgctgcg gctgaagcag aggcgccgca cgctgaagaa ccgcggctac 961 gcgcaggcct gtcgctccaa gcggctgcag cagcggcgcg ggctggaggc cgagcgcgcc 1021 cgcctggccg cccagctgga cgcgctgcgg gccgaggtgg cccgcctggc ccgggagcgc

1081 gatctctaca aggctcgctg tgaccggcta acctcgagcg gccccgggtc ^cgggg^acccc

1141 tcccacctct tcctctgagc cgttcagagc accttgtggt gtagtggggg ctgggtgggg

1201 tggctccgcc caggaggcgg ctgcacggtt ctctgcatcg ttaccagagc gccttctggt

1261 cctagccacg ccctgtatga ccgcgcaaat atccccaaag cttttgggtc ctcaagtcat

1321 gcccgaattt agatgctggt cattttctgg agaggggtcc cctcccctta cgaacacaga

1381 aacccagccc acatgactag cacgctgagc tctgcaggga ccagtgccag gcactggggg

1441 gtggaagtgt ggtgacacag tgaatgggag gtggaggagg gttgcagctc ccacctcagt

1501 ttagttttta attcagggtt ttcaacctgt aacacattaa agctgtaatt agcaatgagg

1561 ctgtattttc attctgaagc ttgtaacctc cccattttag cactacagaa ttttcaagat

1621 ttcaatatcc aacaactaga tagattagga cctctatccg agatgctttt tccctgccca

1681 accctgtggc cttcagggct cagagcagca aaggcctgaa gagtgagctc tgggggttgt

1741 tggtgtgggt tgggagagag ctgtgtgcag aagtctggaa acctgggtcc tagtcccagc

1801 tcttccatgg gatccccctg tcaccctgag caaatcagtt gcttcctgga cttgtgttac

1861 ttcatctaat tctcatgtgg attggacgac ttctgctccc tttccagttc tggcatctcc

1921 ccagtatgga agtcccggtg gtctccccaa gaagtcccca agacaatctc gccaaaggca

1981 cctcctatcc tgctgcagtt tcccagctgc agcctaggca ggggatgcac agcccaggcg

2041 aggaagcctg gcttctctgt gagcacatac gtgggtcctc ggcagctccc tccaggctgt

2101 ctgggcctcc agacctgcac agggtgctcc tgccacctcc cacctctctg agggctgagg

2161 tgagacttct cctgggatga caatttgctg agagagtgca gcttttgtga attaaacttg

2221 aagtccaggc agaattctaa tgcaataagc taaatgttct tgcaatttaa gaagtgttca

2281 ttctttatcc ctgcttcagg ctactgtttg tgtgtctgtg tgtgggagag ggagagagag

2341 ggagagagag agagagcaag aaaaagggaa gtccccacct ggagctgggt ggcacatctg

2401 ttatggagag ccaatggaaa tggggtggag gggcaggcat tggaacagat ggatcctccc

2461 cctctcacca ccaccaccac caaactcaga agcctcagtg gcccagctcg gcctgtcagt

2521 ggctatccag gtctcacagt ttccatggta acccagtcct ccagttcagc aggctaaggg

2581 gagggcagat ggggcctctg ggcagagcac aggcgttgtg ctgggagcag ccgcctcacg

2641 agttactgtc atctgtcctc taccggacat aataaatggg agaagctttc gggcatgggg

2701 gaagaggctc ccatatgtcc ctcttccttt ctaccccctc tctccacccc ctccccagct

2761 gtctcttctc ttcccatcac agttcttgct ccctctttcc cctcctctat cccagctcat

2821 ccctcctccg cttaggagcc tgcaatggga gtcctctcct ccatcgctct cagaatttct

2881 ctcgtctcct ctcccttccc caccattggc cactggccat cctctctggc tctggattct

2941 ctctctgagg ggctcacaga acctttagct tctctttctt catccccttc cacactcatt

3001 gttgaaggga gaggatgggg cagggagggt ccttccggga gcccagctct ttttctctca

3061 ctctaaattt cacctgtgtc tatccttgcc tgccctggag ctgtaattca gacccactac

3121 ttttcttgtt cacttttctc acctgcatgg cacacaaaga cagacagaac catccataga

3181 cacctgagca cacaactagg ccacatatac agtaccatac ctgggtctca aaatgatact

3241 cctgggttac ctgagtatac ttaaaaaaac aaagttgcac actgtggtga ttaagagtag

3301 tgctttaggt ggggcgcagt ggttcctgcc tgtaatccca acactttggg aggccaagga

3361 ggaagtttga gaccagcctg ggcaacatag cgagaaccct gtcactacaa aaaaataaaa

3421 taaaataaat tcagatgtaa tggcatgcac ctggagtccc acctacccag gaggctaagg

3481 tggaaggata acttgagccc aggagttcgg ggatgcagtg agccataatc gtgccactgc

3541 attccagcct gggtggcaga gtgagaccct gtttcaagtt gcagttctac cagttttttg 3601 accatgggtg ggttaaaatc aagcctcact tgattgtcag aaaaatggtg aaaataatgg 3661 taaccaccta ctgagaataa atgcatttaa ataagcactt agcataaa

SEQ ID NO: 5 has the following nucleobase sequence (5’-3’)

1 cgccgggggt gtgtacgcca tgagggggag gaagccgaga agcacctccc actgaagccg

61 gcggtaaccg ccccacgcga gaccacccac cagccaggag cggtcgcagg acccgcagcg

121 gcgcgaggcc ttgtttacac cgactgccgc cactgcgttg gccaaccgct cctccgtcac

181 gcgcttcgct cgtcacgcat ctcgcggctt cctggccggc gggctgcaaa cgtcacacga

241 cggccccgcc cccttggcca gccaactgga cagcggagca cgatgattga taggtgcact

301 cctcccagcc cagctccaga atggccctgc cccccagccc cctggccatg gaatatgtca

361 atgactttga cttgatgaag tttgagctgg cagagcggtt ttccgacgcg gcgctggtct

421 cgatgtctgt gcgggagcta aaccggcagc tgcggggctg cgggcgcgac gaggcgctgc

481 ggctgaagca gaggcgccgc acgctgaaga accgcggcta cgcgcaggcc tgtcgctcca

541 agcggctgca gcagcggcgc gggctggagg ccgagcgcgc ccgcctggcc gcccagctgg

601 acgcgctgcg ggccgaggtg gcccgcctgg cccgggagcg cgatctctac aaggctcgct

661 gtgaccggct aacctcgagc ggccccgggt ccggggaccc ctcccacctc ttcctctgag

721 ccgttcagag caccttgtgg tgtagtgggg gctgggtggg gtggctccgc ccaggaggcg

781 gctgcacggt tctctgcatc gttaccagag cgccttctgg tcctagccac gccctgtatg

841 accgcgcaaa tatccccaaa gcttttgggt cctcaagtca tgcccgaatt tagatgctgg

901 tcattttctg gagaggggtc ccctcccctt acgaacacag aaacccagcc cacatgacta

961 gcacgctgag ctctgcaggg accagtgcca ggcactgggg ggtggaagtg tggtgacaca

1021 gtgaatggga ggtggaggag ggttgcagct cccacctcag tttagttttt aattcagggt

1081 tttcaacctg taacacatta aagctgtaat tagcaatgag gctgtatttt cattctgaag

1141 cttgtaacct ccccatttta gcactacaga attttcaaga tttcaatatc caacaactag

1201 atagattagg acctctatcc gagatgcttt ttccctgccc aaccctgtgg ccttcagggc

1261 tcagagcagc aaaggcctga agagtgagct ctgggggttg ttggtgtggg ttgggagaga

1321 gctgtgtgca gaagtctgga aacctgggtc ctagtcccag ctcttccatg ggatccccct

1381 gtcaccctga gcaaatcagt tgcttcctgg acttgtgtta cttcatctaa ttctcatgtg

1441 gattggacga cttctgctcc ctttccagtt ctggcatctc cccagtatgg aagtcccggt

1501 ggtctcccca agaagtcccc aagacaatct cgccaaaggc acctcctatc ctgctgcagt

1561 ttcccagctg cagcctaggc aggggatgca cagcccaggc gaggaagcct ggcttctctg

1621 tgagcacata cgtgggtcct cggcagctcc ctccaggctg tctgggcctc cagacctgca

1681 cagggtgctc ctgccacctc ccacctctct gagggctgag gtgagacttc tcctgggatg

1741 acaatttgct gagagagtgc agcttttgtg aattaaactt gaagtccagg cagaattcta

1801 atgcaataag ctaaatgttc ttgcaattta agaagtgttc attctttatc cctgcttcag

1861 gctactgttt gtgtgtctgt gtgtgggaga gggagagaga gggagagaga gagagagcaa

1921 gaaaaaggga agtccccacc tggagctggg tggcacatct gttatggaga gccaatggaa

1981 atggggtgga ggggcaggca ttggaacaga tggatcctcc ccctctcacc accaccacca

2041 ccaaactcag aagcctcagt ggcccagctc ggcctgtcag tggctatcca ggtctcacag

2101 tttccatggt aacccagtcc tccagttcag caggctaagg ggagggcaga tggggcctct

2161 gggcagagca caggcgttgt gctgggagca gccgcctcac gagttactgt catctgtcct

2221 ctaccggaca taataaatgg gagaagcttt cgggcatggg ggaagaggct cccatatgtc

2281 cctcttcctt tctaccccct ctctccaccc cctccccagc tgtctcttct cttcccatca 2341 cagttcttgc tccctctttc ccctcctcta tcccagctca tccctcctcc gcttaggagc

2401 ctgcaatggg agtcctctcc tccatcgctc tcagaatttc tctcgtctcc tctcccttcc

2461 ccaccattgg ccactggcca tcctctctgg ctctggattc tctctctgag gggctcacag

2521 aacctttagc ttctctttct tcatcccctt ccacactcat tgttgaaggg agaggatggg

2581 gcagggaggg tccttccggg agcccagctc tttttctctc actctaaatt tcacctgtgt

2641 ctatccttgc ctgccctgga gctgtaattc agacccacta cttttcttgt tcacttttct

2701 cacctgcatg gcacacaaag acagacagaa ccatccatag acacctgagc acacaactag

2761 gccacatata cagtaccata cctgggtctc aaaatgatac tcctgggtta cctgagtata

2821 cttaaaaaaa caaagttgca cactgtggtg attaagagta gtgctttagg tggggcgcag

2881 tggttcctgc ctgtaatccc aacactttgg gaggccaagg aggaagtttg agaccagcct

2941 gggcaacata gcgagaaccc tgtcactaca aaaaaataaa ataaaataaa ttcagatgta

3001 atggcatgca cctggagtcc cacctaccca ggaggctaag gtggaaggat aacttgagcc

3061 caggagttcg gggatgcagt gagccataat cgtgccactg cattccagcc tgggtggcag

3121 agtgagaccc tgtttcaagt tgcagttcta ccagtttttt gaccatgggt gggttaaaat

3181 caagcctcac ttgattgtca gaaaaatggt gaaaataatg gtaaccacct actgagaata

3241 aatgcattta aataagcact tagcataaa

The term “nucleobase,” as used herein, represents a nitrogen-containing heterocyclic ring found at the T position of the ribofuranose/2’-deoxyribofuranose of a nucleoside. Nucleobases are unmodified or modified. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6 substituted purines, as well as synthetic and natural nucleobases, e.g., 5-methylcytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl) adenine and guanine, 2-alkyl (e.g., 2-propyl) adenine and guanine, 2-thiouracil, 2- thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 5- trifluoromethyl uracil, 5-trifluoromethyl cytosine, 7-methyl guanine, 7-methyl adenine, 8-azaguanine, 8- azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine. Certain nucleobases are particularly useful for increasing the binding affinity of nucleic acids, e g., 5-substituted pyrimidines; 6- azapyrimidines; N2-, N6-, and/or 06-substituted purines. Nucleic acid duplex stability can be enhanced using, e.g., 5-methylcytosine. Non-limiting examples of nucleobases include: 2-aminopropyladenine, 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N- methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl ( — CºC — CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7- methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3- deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5- methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1 ,3-diazaphenoxazine-2-one, 1 ,3-diazaphenothiazine-2-one and 9-(2- aminoethoxy)-1 ,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deazaadenine, 7- deazaguanine, 2-aminopyridine, or 2-pyridone. Further nucleobases include those disclosed in Merigan et al., U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991 , 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press, 2008, 163-166 and 442- 443.

The term “nucleoside,” as used herein, represents sugar-nucleobase compounds and groups known in the art, as well as modified or unmodified 2’-deoxyribofuranose-nucleobase compounds and groups known in the art. The sugar may be ribofuranose. The sugar may be modified or unmodified.

An unmodified ribofuranose-nucleobase is ribofuranose having an anomeric carbon bond to an unmodified nucleobase. Unmodified ribofuranose-nucleobases are adenosine, cytidine, guanosine, and uridine. Unmodified 2’-deoxyribofuranose-nucleobase compounds are 2’-deoxyadenosine, 2’- deoxycytidine, 2’-deoxyguanosine, and thymidine. The modified compounds and groups include one or more modifications selected from the group consisting of nucleobase modifications and sugar modifications described herein. A nucleobase modification is a replacement of an unmodified nucleobase with a modified nucleobase. A sugar modification may be, e.g., a 2’-substitution, locking, carbocyclization, or unlocking. A 2’-substitution is a replacement of 2’-hydroxyl in ribofuranose with 2’- fluoro, 2’-methoxy, or 2’-(2-methoxy)ethoxy. Alternatively, a 2’-substitution may be a 2’-(ara) substitution, which corresponds to the following structure:

where B is a nucleobase, and R is a 2’-(ara) substituent (e.g., fluoro). 2’-(ara) substituents are known in the art and can be same as other 2’-substituents described herein. In some embodiments, 2’-(ara) substituent is a 2’-(ara)-F substituent (R is fluoro). A locking modification is an incorporation of a bridge between 4’-carbon atom and 2’-carbon atom of ribofuranose. Nucleosides having a locking modification are known in the art as bridged nucleic acids, e.g., locked nucleic acids (LNA), ethylene-bridged nucleic acids (ENA), and cEt nucleic acids. The bridged nucleic acids are typically used as affinity enhancing nucleosides.

The term “nucleotide,” as used herein, represents a nucleoside bonded to an internucleoside linkage or a monovalent group of the following structure -X¹-P(X²)(R¹)2, where X¹ is O, S, or NH, and X² is absent, =0, or =S, and each R¹ is independently -OH, -N(R²)2, or-0-CH₂CH₂CN, where each R² is independently an optionally substituted alkyl, or both R² groups, together with the nitrogen atom to which they are attached, combine to form an optionally substituted heterocyclyl.

The term “oligonucleotide,” as used herein, represents a structure containing 10 or more contiguous nucleosides covalently bound together by internucleoside linkages. An oligonucleotide includes a 5’ end and a 3’ end. The 5’ end of an oligonucleotide may be, e.g., hydroxyl, a hydrophobic moiety, 5’ cap, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, diphosphrodithioate, triphosphorodithioate, phosphonate, phosphoramidate, a cell penetrating peptide, an endosomal escape moiety, or a neutral organic polymer. The 3’ end of an oligonucleotide may be, e.g., hydroxyl, a hydrophobic moiety, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, disphorodithioate, triphosphorodithioate, phosphonate, phosphoramidate, a cell penetrating peptide, an endosomal escape moiety, or a neutral organic polymer (e.g., polyethylene glycol). An oligonucleotide having a 5’-hydroxyl or 5’-phosphate has an unmodified 5’ terminus. An oligonucleotide having a 5’ terminus other than 5’-hydroxyl or 5’-phosphate has a modified 5’ terminus. An oligonucleotide having a 3’-hydroxyl or 3’-phosphate has an unmodified 3’ terminus. An oligonucleotide having a 3’ terminus other than 3’-hydroxyl or 3’-phosphate has a modified 3’ terminus. Oligonucleotides can be in double- or single- stranded form. Double-stranded oligonucleotide molecules can optionally include one or more single- stranded segments (e.g., overhangs).

All references to “oligonucleotides” include free base forms and also to salts and/or solvates thereof, including pharmaceutically acceptable salts and/or solvates thereof, unless otherwise indicated

The term “oxo,” as used herein, represents a divalent oxygen atom (e.g., the structure of oxo may be shown as =0).

The term “pharmaceutically acceptable,” as used herein, refers to those compounds, materials, compositions, and/or dosage forms, which are suitable for contact with the tissues of an individual (e.g., a human), without excessive toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit/risk ratio.

The term “pharmaceutical composition,” as used herein, represents a composition containing an oligonucleotide described herein, formulated with a pharmaceutically acceptable excipient, and manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a subject.

The term “pharmaceutically acceptable salt,” as used herein, means any pharmaceutically acceptable salt of a compound (e.g., an oligonucleotide) disclosed herein. Pharmaceutically acceptable salts of any of the compounds described herein may include those that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting a free base group with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

The term “protecting group,” as used herein, represents a group intended to protect a functional group (e.g., a hydroxyl, an amino, or a carbonyl) from participating in one or more undesirable reactions during chemical synthesis. The term “O-protecting group,” as used herein, represents a group intended to protect an oxygen containing (e.g., phenol, hydroxyl or carbonyl) group from participating in one or more undesirable reactions during chemical synthesis. The term “/V-protecting group,” as used herein, represents a group intended to protect a nitrogen containing (e.g., an amino or hydrazine) group from participating in one or more undesirable reactions during chemical synthesis. Commonly used O- and N- protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” 3^rd Edition (John Wiley & Sons, New York, 1999), which is incorporated herein by reference. Exemplary O- and N- protecting groups include alkanoyl, aryloyl, or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, ochlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, f-butyldimethylsilyl, tri-/so- propylsilyloxymethyl, 4,4'-dimethoxytrityl, isobutyryl, phenoxyacetyl, 4-isopropylpehenoxyacetyl, dimethylformamidino, and 4-nitrobenzoyl.

Exemplary O-protecting groups for protecting carbonyl containing groups include, but are not limited to: acetals, acylals, 1 ,3-dithianes, 1 ,3-dioxanes, 1 ,3-dioxolanes, and 1 ,3-dithiolanes.

Other O-protecting groups include, but are not limited to: substituted alkyl, aryl, and arylalkyl ethers (e.g., trityl; methylthiomethyl; methoxy methyl; benzyloxymethyl; siloxymethyl; 2,2,2,- trichloroethoxymethyl; tetrahydropyranyl; tetrahydrofuranyl; ethoxyethyl; 1-[2-(trimethylsilyl)ethoxy]ethyl; 2-trimethylsilylethyl; t-butyl ether; p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, benzyl, p- methoxybenzyl, and nitrobenzyl); silyl ethers (e.g., trimethylsilyl; triethylsilyl; triisopropylsilyl; dimethylisopropylsilyl; t-butyldimethylsilyl; t-butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; and diphenymethylsilyl); carbonates (e.g., methyl, methoxymethyl, 9-fluorenylmethyl; ethyl; 2,2,2- trichloroethyl; 2-(trimethylsilyl)ethyl; vinyl, allyl, nitrophenyl; benzyl; methoxybenzyl; 3,4-dimethoxybenzyl; and nitrobenzyl).

Other /V-protecting groups include, but are not limited to, chiral auxiliaries such as protected or unprotected D, L or D, L-amino acids such as alanine, leucine, phenylalanine, and the like; sulfonyl- containing groups such as benzenesulfonyl, p-toluenesulfonyl, and the like; carbamate forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p- nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4- dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyl oxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,

3.4.5-trimethoxybenzyloxycarbonyl, 1 -(p-biphenylyl)-l -methylethoxycarbonyl, a,a-dimethyl-

3.5-dimethoxybenzyloxycarbonyl, benzhydroxy carbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropoxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and the like, arylalkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl, and the like and silyl groups such as trimethylsilyl, and the like. The term “skipmer,” as used herein, refers a gapmer, in which alternating internucleoside linkages are phosphate phosphodiester linkages and intervening internucleoside linkages are modified internucleoside linkages.

The term “stereochemically enriched,” as used herein, refers to a local stereochemical preference for one enantiomer of the recited group over the opposite enantiomer of the same group. Thus, an oligonucleotide containing a stereochemically enriched internucleoside linkage is an oligonucleotide, in which a phosphorothioate of predetermined stereochemistry is present in preference to a phosphorothioate of stereochemistry that is opposite of the predetermined stereochemistry. This preference can be expressed numerically using a diastereomeric ratio for the phosphorothioate of the predetermined stereochemistry. The diastereomeric ratio for the phosphorothioate of the predetermined stereochemistry is the molar ratio of the diastereomers having the identified phosphorothioate with the predetermined stereochemistry relative to the diastereomers having the identified phosphorothioate with the stereochemistry that is opposite of the predetermined stereochemistry. The diastereomeric ratio for the phosphorothioate of the predetermined stereochemistry may be greater than or equal to 1.1 (e.g., greater than or equal to 4, greater than or equal to 9, greater than or equal to 19, or greater than or equal to 39).

The term “subject,” as used herein, represents a human or non-human animal (e.g., a mammal) that is suffering from, or is at risk of, disease, disorder, or condition, as determined by a qualified professional (e.g., a doctor or a nurse practitioner) with or without known in the art laboratory test(s) of sample(s) from the subject. Non-limiting examples of diseases, disorders, and conditions include retinitis pigmentosa (e.g., Rho P23H-associated retinitis pigmentosa, PDE6-associated retinitis pigmentosa, MERTK-associated retinitis pigmentosa, BBS1 -associated retinitis pigmentosa, Rho-associated retinitis pigmentosa, MRFP-associated retinitis pigmentosa, RLBP1 -associated retinitis pigmentosa, RP1- associated retinitis pigmentosa, RPGR-X-linked retinitis pigmentosa, NR2E3-associated retinitis pigmentosa, or SPATA7-associated retinitis pigmentosa), Stargardt disease (e.g., ABCA4-associated Stargardt disease), cone-rod dystrophy (e.g., AIPL1 -associated cone-rod dystrophy or RGRIP1- associated cone-rod dystrophy), Leber congenital amaurosis (e.g., AIPL1 -associated Leber congenital amaurosis, GUCY2D-associated Leber congenital amaurosis, RD3-associated Leber congenital amaurosis, RPE65-associated Leber congenital amaurosis, or SPATA7-associated Leber congenital amaurosis), Bardet Biedl syndrome (e.g., BBS1 -associated Bardet Biedl syndrome), macular dystrophy (e.g., BEST1 -associated macular dystrophy), dry macular degeneration, geographic atrophy, atrophic age-related macular degeneration (AMD), advanced dry AMD, retinal dystrophy (e.g., CEP290- associated retinal dystrophy, CDH3-associated retinal dystrophy, CRB1 -associated retinal dystrophy, or PRPH2-associated retinal dystrophy), choroideremia (e.g., CHM-associated choroideremia), Usher syndrome type 1 (e.g., MY07A-associated Usher syndrome), retinoschisis (e.g., RS1-X-linked retinoschisis), Leber hereditary optic neuropathy (e.g., ND4-associated Lebe’rs hereditary optic neuropathy), and achromatopsia (e.g., CNGA3-associated achromatopsia or CNGB3-associated achromatopsia). Non-limiting examples of diseases, disorders, and conditions include ocular diseases, disorders, and conditions associated with a dysfunction of ABCA4, AIPL1 , BBS1 , BEST1 , CEP290,

CDH3, CHM, CNGA3, CNGB3, CRB1 , GUCY2D, MERTK, MRFP, MY07A, ND4, NR2E3, PDE6, PRPH2, RD3, RHO, RLBP1 , RP1 , RPE65, RPGR, RPGRIP1 , RS1 , or SPATA7 gene. A “sugar” or “sugar moiety,” includes naturally occurring sugars having a furanose ring or a structure that is capable of replacing the furanose ring of a nucleoside. Sugars included in the nucleosides of the invention may be non-furanose (or 4'-substituted furanose) rings or ring systems or open systems. Such structures include simple changes relative to the natural furanose ring (e.g., a six- membered ring). Alternative sugars may also include sugar surrogates wherein the furanose ring has been replaced with another ring system such as, e.g., a morpholino or hexitol ring system. Non-limiting examples of sugar moieties useful that may be included in the oligonucleotides of the invention include b- D-ribose, p-D-2'-deoxyribose, substituted sugars (e.g., 2', 5', and bis substituted sugars), 4'-S-sugars (e.g., 4'-S-ribose, 4'-S-2'-deoxyribose, and 4'-S-2'-substituted ribose), bicyclic sugar moieties (e.g., the 2'- O — CH₂-4' or2'-0 — (CH₂)₂-4' bridged ribose derived bicyclic sugars) and sugar surrogates (when the ribose ring has been replaced with a morpholino or a hexitol ring system).

The term “tailmer,” as used herein, refers to an oligonucleotide having an RNase H recruiting region (gap) flanked by a 3’ wing including at least one affinity enhancing nucleoside (e.g., 1 , 2, 3, or 4 affinity enhancing nucleosides).

“Treatment” and "treating," as used herein, refer to the medical management of a subject with the intent to improve, ameliorate, stabilize, or prevent a disease, disorder, or condition (e.g., retinitis pigmentosa). This term includes active treatment (treatment directed to improve retinitis pigmentosa); causal treatment (treatment directed to the cause of associated retinitis pigmentosa); palliative treatment (treatment designed for the relief of symptoms of retinitis pigmentosa); preventative treatment (treatment directed to minimizing or partially or completely inhibiting the development of retinitis pigmentosa); and supportive treatment (treatment employed to supplement another therapy).

The term “unimer,” as used herein, refers to an oligonucleotide having a pattern of structural features characterized by all of the internucleoside linkages having the same structural feature. By same structural feature is meant the same stereochemistry at the internucleoside linkage phosphorus or the same modification at the linkage phosphorus. In instances, where the “same structural feature” refers to the stereochemical configuration of the internucleoside linkages, the unimer is a “stereounimer.”

Enumeration of positions within oligonucleotides and nucleic acids, as used herein and unless specified otherwise, starts with the 5’-terminal nucleoside as 1 and proceeds in the 3’-direction.

The compounds described herein, unless otherwise noted, encompass isotopically enriched compounds (e.g., deuterated compounds), tautomers, and all stereoisomers and conformers (e.g. enantiomers, diastereomers, E/Z isomers, atropisomers, etc.), as well as racemates thereof and mixtures of different proportions of enantiomers or diastereomers, or mixtures of any of the foregoing forms as well as salts (e.g., pharmaceutically acceptable salts).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a bar graph quantifying the effect of an Nrl targeting ASO upon rod and cone gene expression. The data were normalized to negative scrambled control. Treatment with the Nrl-targeting ASO decreased Nrl and rod gene expression without decreasing cone gene expression or the expression of Otx2. Otx2 is an upstream transcription factor expressed in both rod and cone photoreceptors.

FIGS. 2A-2J show dosage curves quantifying the mRNA inhibitory effect of the indicated Nrl- targeting oligonucleotides in HEK293 cells expressing high levels of the target homo sapiens (GenBank ID: BC012395.1). The data were normalized to GFP and/or actin and are represented as a percentage of a negative scrambled control. Treatment with Nrl-targeting oligonucleotides resulted in a dose-dependent decrease in Nrl gene expression, relative to the vehicle (DPBS) control. FIG. 2A shows dosage curves for chemically modified version of SEQ ID NO: 7. FIG. 2B shows dosage curves for chemically modified version of SEQ ID NO: 9. FIG. 2C shows dosage curves for chemically modified version of SEQ ID NO:

10. FIG. 2D shows dosage curves for chemically modified version of SEQ ID NO: 11. FIG. 2E shows dosage curves for chemically modified version of SEQ ID NO: 12. FIG. 2F shows dosage curves for chemically modified version of SEQ ID NO: 13. FIG. 2G shows dosage curves for chemically modified version of SEQ ID NO: 14. FIG. 2H shows dosage curves for chemically modified version of SEQ ID NO: 15. FIG. 2I shows dosage curves for chemically modified version of SEQ ID NO: 16. FIG. 2J shows dosage curves for chemically modified version of SEQ ID NO: 17. (See Table 1 , below, regarding chemically modified versions of the sequences described herein.)

FIGS. 3A-3E show dosage curves quantifying the mRNA inhibitory effect of the indicated Nrl- targeting oligonucleotides in HEK293 cells expressing high levels of the target Macaca fascicularis (GenBank ID: XM_015453620.1). The data were normalized to GFP and/or actin and are represented as a percentage of a vehicle (DPBS) control. Treatment with Nrl-targeting oligonucleotides resulted in a dose-dependent decrease in Nrl gene expression, relative to the negative scrambled control. FIG. 3A shows dosage curves for chemically modified version of SEQ ID NO: 9. FIG. 3B shows dosage curves for chemically modified version of SEQ ID NO: 10. FIG. 3C shows dosage curves for chemically modified version of SEQ ID NO: 14. FIG. 3D shows dosage curves for chemically modified version of SEQ ID NO: 15. FIG. 3E shows dosage curves for chemically modified version of SEQ ID NO: 16. (See Table l , below, regarding chemically modified versions of the sequences described herein.)

DETAILED DESCRIPTION

In general, the invention provides oligonucleotides that may be used in the treatment of ocular degeneration disorders (e.g., a retinal degeneration disorder; e.g., retinitis pigmentosa (e.g., Rho P23H- associated retinitis pigmentosa, PDE6-associated retinitis pigmentosa, MERTK-associated retinitis pigmentosa, BBS1 -associated retinitis pigmentosa, Rho-associated retinitis pigmentosa, MRFP- associated retinitis pigmentosa, RLBP1 -associated retinitis pigmentosa, RP1 -associated retinitis pigmentosa, RPGR-X-linked retinitis pigmentosa, NR2E3-associated retinitis pigmentosa, or SPATA7- associated retinitis pigmentosa), Stargardt disease (e.g., ABCA4-associated Stargardt disease), cone-rod dystrophy (e.g., AIPL1 -associated cone-rod dystrophy or RGRIP1 -associated cone-rod dystrophy), Leber congenital amaurosis (e.g., AIPL1 -associated Leber congenital amaurosis, GUCY2D-associated Leber congenital amaurosis, RD3-associated Leber congenital amaurosis, RPE65-associated Leber congenital amaurosis, or SPATA7-associated Leber congenital amaurosis), Bardet Biedl syndrome (e.g., BBS1- associated Bardet Biedl syndrome), macular dystrophy (e.g., BEST1 -associated macular dystrophy), dry macular degeneration, geographic atrophy, atrophic age-related macular degeneration (AMD), advanced dry AMD, retinal dystrophy (e.g., CEP290-associated retinal dystrophy, CDH3-associated retinal dystrophy, CRB1 -associated retinal dystrophy, or PRPH2-associated retinal dystrophy), choroideremia (e.g., CHM-associated choroideremia), Usher syndrome type 1 (e.g., MY07A-associated Usher syndrome), retinoschisis (e.g., RS1-X-linked retinoschisis), Leber hereditary optic neuropathy (e.g., ND4- associated Lebe’rs hereditary optic neuropathy), and achromatopsia (e.g., CNGA3-associated achromatopsia or CNGB3-associated achromatopsia)). Without wishing to be bound by theory, reduction of the expression of NRL in photoreceptor cells can prevent loss of photoreceptor cells, thereby treating an ocular degeneration disorder (e.g., a retinal degeneration disorder).

The invention provides an antisense approach to reducing expression of NRL in cells as described herein. Typically, an antisense activity may be observed directly or indirectly. Observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid, a change in the ratio of splice variants of a nucleic acid or protein, and/or a phenotypic change in a cell or animal.

I. Antisense

In one approach, the invention provides a single-stranded oligonucleotide having a nucleobase sequence with at least 6 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 or a modified version thereof (e.g., SEQ ID NOs: 18-29). In some embodiments, the single-stranded oligonucleotide may be delivered to a patient as a double stranded oligonucleotide, where the oligonucleotide of the invention is hybridized to another oligonucleotide (e.g., an oligonucleotide having a total of 12 to 30 nucleotides).

An antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) includes a nucleobase sequence having at least 6 (e.g., at least 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16) contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 or a modified version thereof (e.g., SEQ ID NOs: 18-29). For example, the oligonucleotide may include a total of at least 7 (e.g., at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, or at least 16) interlinked nucleotides, e.g., the oligonucleotide may include a total a total of 25 interlinked nucleotides or fewer (e.g., 20 interlinked nucleotides or fewer, 19 interlinked nucleotides or fewer, 18 interlinked nucleotides or fewer, 17 interlinked nucleotides or fewer, 16 interlinked nucleotides or fewer, 15 interlinked nucleotides or fewer, 14 interlinked nucleotides or fewer,

13 interlinked nucleotides or fewer, 12 interlinked nucleotides or fewer, or 11 interlinked nucleotides or fewer). Thus, the single-stranded oligonucleotide may include, e.g., at least 8 (e.g., at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, or at least 16) contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 or a modified version thereof (e.g., SEQ ID NOs: 18-29).

An antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) may be a gapmer, headmer, ortailmer. Gapmers are oligonucleotides having an RNase H recruiting region (gap) flanked by a 5' wing and 3' wing, each of the wings including at least one affinity enhancing nucleoside (e.g., 1, 2, 3, or 4 affinity enhancing nucleosides). Headmers are oligonucleotides having an RNase H recruiting region (gap) flanked by a 5’ wing including at least one affinity enhancing nucleoside (e.g., 1 , 2, 3, or 4 affinity enhancing nucleosides). Tailmers are oligonucleotides having an RNase H recruiting region (gap) flanked by a 3’ wing including at least one affinity enhancing nucleoside (e.g., 1 , 2, 3, or 4 affinity enhancing nucleosides). In certain embodiments, each wing includes 1-5 nucleosides. In some embodiments, each nucleoside of each wing is a modified nucleoside. In particular embodiments, the gap includes 7-12 nucleosides. Typically, the gap region includes a plurality of contiguous, unmodified deoxyribonucleotides. For example, all nucleotides in the gap region are unmodified deoxyribonucleotides (2’-deoxyribofuranose-based nucleotides). Preferably, an antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) is a gapmerwith 30-38% of the nucleotides being LNAs.

The 5'-wing may consists of, e.g., 1 to 8 nucleosides. The 5'-wing may consist of, e.g., 1 to 7 nucleosides. The 5'-wing may consist of, e.g., 1 to 6 linked nucleosides. The 5'-wing may consist of, e.g., 1 to 5 linked nucleosides. The 5'-wing may consist of, e.g., 2 to 5 linked nucleosides. The 5'-wing may consist of, e.g., 3 to 5 linked nucleosides. The 5'-wing may consist of, e.g., 4 or 5 linked nucleosides. The 5'-wing may consist of, e.g., 1 to 4 linked nucleosides. The 5'-wing may consist of, e.g., 1 to 3 linked nucleosides. The 5'-wing may consist of, e.g., 1 or 2 linked nucleosides. The 5'-wing may consist of, e.g., 2 to 4 linked nucleosides. The 5'-wing may consist of, e.g., 2 or 3 linked nucleosides. The 5'-wing may consist of, e.g., 3 or 4 linked nucleosides. The 5'-wing may consist of, e.g., 1 nucleoside. The 5'-wing may consist of, e.g., 2 linked nucleosides. The 5'-wing may consist of, e.g., 3 linked nucleosides. The 5'-wing may consist of, e.g., 4 linked nucleosides. The 5'-wing may consist of, e.g., 5 linked nucleosides. The 5'-wing may consist of, e.g., 6 linked nucleosides.

The 3’-wing may consists of, e.g., 1 to 8 nucleosides. The 3’-wing may consist of, e.g., 1 to 7 nucleosides. The 3’-wing may consist of, e.g., 1 to 6 linked nucleosides. The 3’-wing may consist of, e.g., 1 to 5 linked nucleosides. The 3’-wing may consist of, e.g., 2 to 5 linked nucleosides. The 3’-wing may consist of, e.g., 3 to 5 linked nucleosides. The 3’-wing may consist of, e.g., 4 or 5 linked nucleosides. The 3’-wing may consist of, e.g., 1 to 4 linked nucleosides. The 3’-wing may consist of, e.g., 1 to 3 linked nucleosides. The 3’-wing may consist of, e.g., 1 or 2 linked nucleosides. The 3’-wing may consist of, e.g., 2 to 4 linked nucleosides. The 3’-wing may consist of, e.g., 2 or 3 linked nucleosides. The 3’-wing may consist of, e.g., 3 or 4 linked nucleosides. The 3’-wing may consist of, e.g., 1 nucleoside. The 3’-wing may consist of, e.g., 2 linked nucleosides. The 3’-wing may consist of, e.g., 3 linked nucleosides. The 3’-wing may consist of, e.g., 4 linked nucleosides. The 3’-wing may consist of, e.g., 5 linked nucleosides. The 3’-wing may consist of, e.g., 6 linked nucleosides.

The 5'-wing may include, e.g., at least one bridged nucleoside. The 5'-wing may include, e.g., at least two bridged nucleosides. The 5'-wing may include, e.g., at least three bridged nucleosides. The 5'- wing may include, e.g., at least four bridged nucleosides. The 5'-wing may include, e.g., at least one constrained ethyl (cEt) nucleoside. The 5'-wing may include, e.g., at least one LNA nucleoside. Each nucleoside of the 5'-wing may be, e.g., a bridged nucleoside. Each nucleoside of the 5'-wing may be, e.g., a constrained ethyl (cEt) nucleoside. Each nucleoside of the 5'-wing may be, e.g., a LNA nucleoside.

The 3’-wing may include, e.g., at least one bridged nucleoside. The 3’-wing may include, e.g., at least two bridged nucleosides. The 3’-wing may include, e.g., at least three bridged nucleosides. The 3’- wing may include, e.g., at least four bridged nucleosides. The 3’-wing may include, e.g., at least one constrained ethyl (cEt) nucleoside. The 3’-wing may include, e.g., at least one LNA nucleoside. Each nucleoside of the 3’-wing may be, e.g., a bridged nucleoside. Each nucleoside of the 3’-wing may be, e.g., a constrained ethyl (cEt) nucleoside. Each nucleoside of the 3’-wing may be, e.g., a LNA nucleoside.

The 5'-wing may include, e.g., at least one non-bicyclic modified nucleoside. The 5'-wing may include, e.g., at least one 2'-substituted nucleoside. The 5'-wing may include, e.g., at least one 2'-MOE nucleoside. The 5'-wing may include, e.g., at least one 2'-OMe nucleoside. Each nucleoside of the 5'- wing may be, e.g., a non-bicyclic modified nucleoside. Each nucleoside of the 5'-wing may be, e.g., a 2'- substituted nucleoside. Each nucleoside of the 5'-wing may be, e.g., a 2'-MOE nucleoside. Each nucleoside of the 5'-wing may be, e.g., a 2'-OMe nucleoside.

The 3’-wing may include, e.g., at least one non-bicyclic modified nucleoside. The 3’-wing may include, e.g., at least one 2'-substituted nucleoside. The 3’-wing may include, e.g., at least one 2'-MOE nucleoside. The 3’-wing may include, e.g., at least one 2'-OMe nucleoside. Each nucleoside of the 3’- wing may be, e.g., a non-bicyclic modified nucleoside. Each nucleoside of the 3’-wing may be, e.g., a 2'- substituted nucleoside. Each nucleoside of the 3’-wing may be, e.g., a 2'-MOE nucleoside. Each nucleoside of the 3’-wing may be, e.g., a 2'-OMe nucleoside.

The gap may consist of, e.g., 6 to 20 linked nucleosides. The gap may consist of, e.g., 6 to 15 linked nucleosides. The gap may consist of, e.g., 6 to 12 linked nucleosides. The gap may consist of, e.g., 6 to 10 linked nucleosides. The gap may consist of, e.g., 6 to 9 linked nucleosides. The gap may consist of, e.g., 6 to 8 linked nucleosides. The gap may consist of, e.g., 6 or 7 linked nucleosides. The gap may consist of, e.g., 7 to 10 linked nucleosides. The gap may consist of, e.g., 7 to 9 linked nucleosides. The gap may consist of, e.g., 7 or 8 linked nucleosides. The gap may consist of, e.g., 8 to 10 linked nucleosides. The gap may consist of, e.g., 8 or 9 linked nucleosides. The gap may consist of, e.g., 6 linked nucleosides. The gap may consist of, e.g., 7 linked nucleosides. The gap may consist of, e.g., 8 linked nucleosides. The gap may consist of, e.g., 9 linked nucleosides. The gap may consist of, e.g., 10 linked nucleosides. The gap may consist of, e.g., 11 linked nucleosides. The gap may consist of, e.g., 12 linked nucleosides.

Each nucleoside of the gap may be, e.g., a 2'-deoxynucleoside. The gap may include, e.g., one or more modified nucleosides. Each nucleoside of the gap may be, e.g., a 2'-deoxynucleoside or may be, e.g., a modified nucleoside that is “DNA-like.” In such embodiments, “DNA-like” means that the nucleoside has similar characteristics to DNA, such that a duplex including the gapmer and an RNA molecule is capable of activating RNase H. For example, under certain conditions, 2'-(ara)-F may support RNase H activation, and thus is DNA-like. In certain embodiments, one or more nucleosides of the gap is not a 2'-deoxynucleoside and is not DNA-like. In certain such embodiments, the gapmer nonetheless supports RNase H activation (e.g., by virtue of the number or placement of the non-DNA nucleosides).

In certain embodiments, gaps include a stretch of unmodified 2'-deoxynucleoside interrupted by one or more modified nucleosides, thus resulting in three sub-regions (two stretches of one or more 2'- deoxynucleosides and a stretch of one or more interrupting modified nucleosides). In certain embodiments, no stretch of unmodified 2'-deoxynucleosides is longer than 5, 6, or 7 nucleosides. In certain embodiments, such short stretches is achieved by using short gap regions. In certain embodiments, short stretches are achieved by interrupting a longer gap region.

The gap may include, e.g., one or more modified nucleosides. The gap may include, e.g., one or more modified nucleosides selected from among cEt, FHNA, LNA, and 2-thio-thymidine. The gap may include, e.g., one modified nucleoside. The gap may include, e.g., a 5'-substituted sugar moiety selected from the group consisting of 5'-Me and 5'-(R)-Me. The gap may include, e.g., two modified nucleosides. The gap may include, e.g., three modified nucleosides. The gap may include, e.g., four modified nucleosides. The gap may include, e.g., two or more modified nucleosides and each modified nucleoside is the same. The gap may include, e.g., two or more modified nucleosides and each modified nucleoside is different. The gap may include, e.g., one or more modified internucleoside linkages. The gap may include, e.g., one or more methyl phosphonate linkages. In certain embodiments the gap may include, e.g., two or more modified internucleoside linkages. The gap may include, e.g., one or more modified linkages and one or more modified nucleosides. The gap may include, e.g., one modified linkage and one modified nucleoside. The gap may include, e.g., two modified linkages and two or more modified nucleosides.

An antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) may include one or more mismatches. For example, the mismatch may be specifically positioned within a gapmer, headmer, ortailmer. The mismatch may be, e.g., at position 1 , 2, 3, 4, 5, 6,

7, or 8 (e.g., at position 1 , 2, 3, or 4) from the 3’-end of the gap region. Alternatively or additionally, the mismatch may be, e.g., at position 9, 8, 7, 6, 5, 4, 3, 2, or 1 (e.g., at position 4, 3, 2, or 1) from the 3'-end of the gap region. In some embodiments, the 5’ wing and/or 3’wing do not include mismatches.

An antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) may be a morpholino.

An antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) may be include a total of X to Y interlinked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number nucleosides in the range. In these embodiments, X and Y are each independently selected from the group consisting of 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X<Y. For example, an oligonucleotide of the invention may include a total of 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21 , 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to

14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21 , 13 to 22, 13 to 23, 13 to 24, 13 to

25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to

20, 14 to 21 , 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to

16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21 , 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to

27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21 , 16 to 22, 16 to 23, 16 to

24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21 , 17 to

22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to

21 , 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to

21 , 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21 , 20 to

22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to

24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to

27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to

25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to

27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 interlinked nucleosides.

In some embodiments, an antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) includes at least one modified internucleoside linkage. A modified internucleoside linkage may be, e.g., a phosphorothioate internucleoside linkage (e.g., a phosphorothioate diester or phosphorothioate triester).

In some embodiments, an antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) includes at least one stereochemically enriched phosphorothioate-based internucleoside linkage. In some embodiments, an antisense oligonucleotide of the invention (e.g., a single-stranded oligonucleotide of the invention) includes a pattern of stereochemically enriched phosphorothioate internucleoside linkages described herein (e.g., a 5’-RpSpSp-3’). These patterns may enhance target NRL nucleic acid cleavage by RNase H relative to a stereorandom corresponding oligonucleotide. In some embodiments, inclusion and/or location of particular stereochemically enriched linkages within an oligonucleotide may alter the cleavage pattern of a target nucleic acid, when such an oligonucleotide is utilized for cleaving the nucleic acid. For example, a pattern of internucleoside linkage P-stereogenic centers may increase cleavage efficiency of a target nucleic acid. A pattern of internucleoside linkage P-stereogenic centers may provide new cleavage sites in a target nucleic acid. A pattern of internucleoside linkage P-stereogenic centers may reduce the number of cleavage sites, for example, by blocking certain existing cleavage sites. Moreover, in some embodiments, a pattern of internucleoside linkage P-stereogenic centers may facilitate cleavage at only one site within the target sequence that is complementary to an oligonucleotide utilized for the cleavage. Cleavage efficiency may be increased by selecting a pattern of internucleoside linkage P-stereogenic centers that reduces the number of cleavage sites in a target nucleic acid.

Purity of an oligonucleotide may be expressed as the percentage of oligonucleotide molecules that are of the same oligonucleotide type within an oligonucleotide composition. At least about 10% of the oligonucleotides may be, e.g., of the same oligonucleotide type. At least about 20% of the oligonucleotides may be, e.g., of the same oligonucleotide type. At least about 30% of the oligonucleotides may be, e.g., of the same oligonucleotide type. At least about 40% of the oligonucleotides may be, e.g., of the same oligonucleotide type. At least about 50% of the oligonucleotides may be, e.g., of the same oligonucleotide type. At least about 60% of the oligonucleotides may be, e.g., of the same oligonucleotide type. At least about 70% of the oligonucleotides may be, e.g., of the same oligonucleotide type. At least about 80% of the oligonucleotides may be, e.g., of the same oligonucleotide type. At least about 90% of the oligonucleotides may be, e.g., of the same oligonucleotide type. At least about 92% of the oligonucleotides may be, e.g., of the same oligonucleotide type. At least about 94% of the oligonucleotides may be, e.g., of the same oligonucleotide type. At least about 95% of the oligonucleotides may be, e.g., of the same oligonucleotide type. At least about 96% of the oligonucleotides may be, e.g., of the same oligonucleotide type. At least about 97% of the oligonucleotides may be, e.g., of the same oligonucleotide type. At least about 98% of the oligonucleotides may be, e.g., of the same oligonucleotide type. At least about 99% of the oligonucleotides may be, e.g., of the same oligonucleotide type.

An oligonucleotide may include one or more stereochemically enriched internucleoside linkages. An oligonucleotide may include two or more stereochemically enriched internucleoside linkages. An oligonucleotide may include three or more stereochemically enriched internucleoside linkages. An oligonucleotide may include four or more stereochemically enriched internucleoside linkages. An oligonucleotide may include five or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 stereochemically enriched internucleoside linkages. An oligonucleotide may include 5 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 6 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 7 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 8 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 9 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 10 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 11 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 12 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 13 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 14 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 15 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 16 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 17 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 18 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 19 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 20 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 21 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 22 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 23 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 24 or more stereochemically enriched internucleoside linkages. An oligonucleotide may include 25 or more stereochemically enriched internucleoside linkages.

An oligonucleotide may include at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% stereochemically enriched internucleoside linkages. Exemplary such stereochemically enriched internucleoside linkages are described herein. An oligonucleotide may include at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% stereochemically enriched internucleoside linkages in the Sp configuration.

A stereochemically enriched internucleoside linkage may be, e.g., a stereochemically enriched phosphorothioate internucleoside linkage. A provided oligonucleotide comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% stereochemically enriched phosphorothioate internucleoside linkages. All stereochemically enriched linkages may be, e.g., stereochemically enriched phosphorothioate internucleoside linkages. At least 10, 20, 30, 40, 50, 60, 70, 80, or 90% stereochemically enriched phosphorothioate internucleoside linkages have the Sp stereochemical configuration. At least 10% stereochemically enriched phosphorothioate internucleoside linkages have the Sp stereochemical configuration. At least 20% stereochemically enriched phosphorothioate internucleoside linkages have the Sp stereochemical configuration. At least 30% stereochemically enriched phosphorothioate internucleoside linkages have the Sp stereochemical configuration. At least 40% stereochemically enriched phosphorothioate internucleoside linkages have the Sp stereochemical configuration. At least 50% stereochemically enriched phosphorothioate internucleoside linkages have the Sp stereochemical configuration. At least 60% stereochemically enriched phosphorothioate internucleoside linkages have the Sp stereochemical configuration. At least 70% stereochemically enriched phosphorothioate internucleoside linkages have the Sp stereochemical configuration. At least 80% stereochemically enriched phosphorothioate internucleoside linkages have the Sp stereochemical configuration. At least 90% stereochemically enriched phosphorothioate internucleoside linkages have the Sp stereochemical configuration. At least 95% stereochemically enriched phosphorothioate internucleoside linkages have the Sp stereochemical configuration. At least 10, 20, 30, 40, 50, 60, 70, 80, or 90% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. At least 10% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. At least 20% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. At least 30% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. At least 40% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. At least 50% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. At least 60% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. At least 70% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. At least 80% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. At least 90% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. At least 95% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. In some embodiments, less than 10, 20, 30, 40, 50, 60, 70, 80, or 90% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. In some embodiments, less than 10% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. In some embodiments, less than 20% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. In some embodiments, less than 30% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. In some embodiments, less than 40% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. In some embodiments, less than 50% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. In some embodiments, less than 60% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. In some embodiments, less than 70% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. In some embodiments, less than 80% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. In some embodiments, less than 90% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. In some embodiments, less than 95% stereochemically enriched phosphorothioate internucleoside linkages have the Rp stereochemical configuration. An oligonucleotide may have, e.g., only one Rp stereochemically enriched phosphorothioate internucleoside linkages. An oligonucleotide may have, e.g., only one Rp stereochemically enriched phosphorothioate internucleoside linkages, where all internucleoside linkages are stereochemically enriched phosphorothioate internucleoside linkages. A stereochemically enriched phosphorothioate internucleoside linkage may be, e.g., a stereochemically enriched phosphorothioate diester linkage. In some embodiments, each stereochemically enriched phosphorothioate internucleoside linkage is independently a stereochemically enriched phosphorothioate diester linkage. In some embodiments, each internucleoside linkage is independently a stereochemically enriched phosphorothioate diester linkage. In some embodiments, each internucleoside linkage is independently a stereochemically enriched phosphorothioate diester linkage, and only one is Rp.

The gap region may include, e.g., a stereochemically enriched internucleoside linkage. The gap region may include, e.g., stereochemically enriched phosphorothioate internucleoside linkages. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is (Sp)mRp or Rp(Sp)m, where m is 2, 3, 4, 5, 6, 7 or 8. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is (Sp)_mRp or Rp(Sp)_m, where m is 2, 3, 4, 5, 6, 7 or 8. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is (Sp)_mRp, where m is 2, 3, 4, 5, 6, 7 or 8. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is Rp(Sp)_m, where m is 2, 3, 4, 5, 6, 7 or 8. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is (Sp)_mRp or Rp(Sp)_m, where m is 2, 3, 4, 5, 6, 7 or 8. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is a motif including at least 33% of internucleoside linkages with the Sp stereochemical identify. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is a motif including at least 50% of internucleoside linkages with the Sp stereochemical identify. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is a motif including at least 66% of internucleoside linkages with the Sp stereochemical identify. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is a repeating triplet motif selected from RpRpSp and SpSpRp. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is a repeating RpRpSp. The gap region may have, e.g., a repeating pattern of internucleoside linkage stereochemistry, where the repeating pattern is a repeating SpSpRp.

An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (Sp)_mRp or Rp(Sp)_m. An oligonucleotide may include a pattern of internucleoside P- stereogenic centers in the gap region including Rp(Sp)_m. An oligonucleotide may include a pattern of P- stereogenic centers in the gap region including (Sp)_mRp. In some embodiments, m is 2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including RP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (SP)2RP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (RP)2RP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including RPSPRP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including SPRPRP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including ( SP)2RP

An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (Sp)mRp or Rp(Sp)m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including Rp(Sp)_m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (Sp)_mRp. In some embodiments, m is 2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including RP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (SP)2RP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (RP)2RP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including RPSPRP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including SPRPRP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (SP)2RP.

In the embodiments of internucleoside P-stereogenic center patterns, m is 2, 3, 4, 5, 6, 7 or 8, unless specified otherwise. In some embodiments of internucleoside P-stereogenic center patterns, m is

3, 4, 5, 6, 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, m is 4, 5, 6, 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, m is 5, 6, 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, m is 6, 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, m is 7 or 8. In some embodiments of internucleoside P- stereogenic center patterns, m is 2. In some embodiments of internucleoside P-stereogenic center patterns, m is 3. In some embodiments of internucleoside P-stereogenic center patterns, m is 4. In some embodiments of internucleoside P-stereogenic center patterns, m is 5. In some embodiments of internucleoside P-stereogenic center patterns, m is 6. In some embodiments of internucleoside P- stereogenic center patterns, m is 7. In some embodiments of internucleoside P-stereogenic center patterns, m is 8.

A repeating pattern may be, e.g., (Sp)_m(Rp)n, where n is independently 1 , 2, 3, 4, 5, 6, 7 or 8, and m is independently as described herein. An oligonucleotide may include a pattern of internucleoside P- stereogenic centers including (Sp)_m(Rp)n. An oligonucleotide may include a pattern of internucleoside P- stereogenic centers including (Sp)m(Rp)n. A repeating pattern may be, e.g., (Rp)_n(Sp)_m, where n is independently 1 , 2, 3, 4, 5, 6, 7 or 8, and m is independently as described herein. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (Rp)_n(Sp)_m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (Rp)_n(Sp)_m. In some embodiments, (Rp)_n(Sp)_m is (RP)(SP)2. In some embodiments, (Sp)_n(Rp)m is (SP)2(RP).

A repeating pattern may be, e.g., (Sp)_m(Rp)n(Sp)t, where each of n and t is independently 1 , 2, 3,

4, 5, 6, 7 or 8, and m is as described herein. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (Sp)_m(Rp)n(Sp)t. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (Sp)_m(Rp)n(Sp)t. A repeating pattern may be, e.g., (Sp)t(Rp)n(Sp)m, where each of n and t is independently 1 , 2, 3, 4, 5, 6, 7 or 8, and m is as described herein. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (Sp)t(Rp)n(Sp)m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (Sp)t(Rp)_n(Sp)_m.

A repeating pattern is (Np)t(Rp)_n(Sp)_m, where each of n and t is independently 1 , 2, 3, 4, 5, 6, 7 or 8, Np is independently Rp or Sp, and m is as described herein. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (Np)t(Rp)_n(Sp)_m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (Np)t(Rp)n(Sp)m. A repeating pattern may be, e.g., (Np)t(Rp)_n(Sp)_m, where each of n and t is independently 1 , 2, 3, 4, 5, 6, 7 or 8, Np is independently Rp or Sp, and m is as described herein. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (Np)t(Rp)_n(Sp)_m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers in the gap region including (Np)t(Rp)_n(Sp)_m. In some embodiments, Np is Rp. In some embodiments, Np is Sp. All Np may be, e.g., same. All Np may be, e.g., Sp. At least one Np may be, e.g., different from another Np. In some embodiments, t is 2.

In the embodiments of internucleoside P-stereogenic center patterns, n is 1 , 2, 3, 4, 5, 6, 7 or 8.

In some embodiments of internucleoside P-stereogenic center patterns, n is 2, 3, 4, 5, 6, 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, n is 3, 4, 5, 6, 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, n is 4, 5, 6, 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, n is 5, 6, 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, n is 6, 7 or 8. In some embodiments of internucleoside P- stereogenic center patterns, n is 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, n is 1. In some embodiments of internucleoside P-stereogenic center patterns, n is 2. In some embodiments of internucleoside P-stereogenic center patterns, n is 3. In some embodiments of internucleoside P-stereogenic center patterns, n is 4. In some embodiments of internucleoside P- stereogenic center patterns, n is 5. In some embodiments of internucleoside P-stereogenic center patterns, n is 6. In some embodiments of internucleoside P-stereogenic center patterns, n is 7. In some embodiments of internucleoside P-stereogenic center patterns, n is 8.

In the embodiments of internucleoside P-stereogenic center patterns, t is 1 , 2, 3, 4, 5, 6, 7 or 8.

In some embodiments of internucleoside P-stereogenic center patterns, t is 2, 3, 4, 5, 6, 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, t is 3, 4, 5, 6, 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, t is 4, 5, 6, 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, t is 5, 6, 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, t is 6, 7 or 8. In some embodiments of internucleoside P- stereogenic center patterns, t is 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, t is 1. In some embodiments of internucleoside P-stereogenic center patterns, t is 2. In some embodiments of internucleoside P-stereogenic center patterns, t is 3. In some embodiments of internucleoside P-stereogenic center patterns, t is 4. In some embodiments of internucleoside P- stereogenic center patterns, t is 5. In some embodiments of internucleoside P-stereogenic center patterns, t is 6. In some embodiments of internucleoside P-stereogenic center patterns, t is 7. In some embodiments of internucleoside P-stereogenic center patterns, t is 8.

At least one of m and t may be, e.g., greater than 2. At least one of m and t may be, e.g., greater than 3. At least one of m and t may be, e.g., greater than 4. At least one of m and t may be, e.g., greater than 5. At least one of m and t may be, e.g., greater than 6. At least one of m and t may be, e.g., greater than 7. In some embodiments, each of m and t is greater than 2. In some embodiments, each of m and t is greater than 3. In some embodiments, each of m and t is greater than 4. In some embodiments, each of m and t is greater than 5. In some embodiments, each of m and t is greater than 6. In some embodiments, each of m and t is greater than 7.

In some embodiments of internucleoside P-stereogenic center patterns, n is 1 , and at least one of m and t is greater than 1 . In some embodiments of internucleoside P-stereogenic center patterns, n is 1 and each of m and t is independent greater than 1. In some embodiments of internucleoside P- stereogenic center patterns, m>n and t>n. In some embodiments, (Sp)_m(Rp)n(Sp)t is (SP)2RP(SP)2. In some embodiments, (Sp)t(Rp)_n(Sp)_m is (SP)2RP(SP)2. In some embodiments, (Sp)t(Rp)_n(Sp)_m is SPRP(SP)2.

In some embodiments, (Np)t(Rp)_n(Sp)_m is (Np)tRp(Sp)_m. In some embodiments, (Np)t(Rp)_n(Sp)_m is (Np)2Rp(Sp)m. In some embodiments, (Np)t(Rp)_n(Sp)_m is (Rp)2Rp(Sp)_m. In some embodiments, (Np)t(Rp)n(Sp)m is (Sp)2Rp(Sp)m. In some embodiments, (Np)t(Rp)_n(Sp)_m is RpSpRp(Sp)_m. In some embodiments, (Np)t(Rp)_n(Sp)_m is SpRpRp(Sp)_m.

In some embodiments, (Sp)t(Rp)_n(Sp)_m is SpRpSpSp. In some embodiments, (Sp)t(Rp)_n(Sp)_m is (SP)2RP(SP)2. In some embodiments, (Sp)t(Rp)_n(Sp)_m is (SP)3RP(SP)3. In some embodiments, (Sp)t(Rp)n(Sp)m is (SP)4RP(SP)4. In some embodiments, (Sp)t(Rp)_n(Sp)_m is (Sp)tRp(Sp)5. In some embodiments, (Sp)t(Rp)_n(Sp)_m is SpRp(Sp)s. In some embodiments, (Sp)t(Rp)_n(Sp)_m is (SP)2RP(SP)5. In some embodiments, (Sp)t(Rp)_n(Sp)_m is (SP)3RP(SP)5. In some embodiments, (Sp)t(Rp)_n(Sp)_m is (SP)4RP(SP)5. In some embodiments, (Sp)t(Rp)_n(Sp)_m is (SP)5RP(SP)5.

In some embodiments, (Sp)_m(Rp)n(Sp)t is (SP)2RP(SP)2. In some embodiments, (Sp)_m(Rp)n(Sp)t is (SP)3RP(SP)3. In some embodiments, (Sp)_m(Rp)n(Sp)t is (SP)4RP(SP)4. In some embodiments, (Sp)m(Rp)n(Sp)t is (Sp)mRp(Sp)5. In some embodiments, (Sp)_m(Rp)n(Sp)t is (SP)2RP(SP)5. In some embodiments, (Sp)_m(Rp)n(Sp)t is (SP)3RP(SP)5. In some embodiments, (Sp)_m(Rp)n(Sp)t is (SP)4RP(SP)5. In some embodiments, (Sp)_m(Rp)n(Sp)t is (SP)5RP(SP)5.

The gap region may include, e.g., at least one Rp internucleoside linkage. The gap region may include, e.g., at least one Rp phosphorothioate internucleoside linkage. The gap region may include, e.g., at least two Rp internucleoside linkages. The gap region may include, e.g., at least two Rp phosphorothioate internucleoside linkages. The gap region may include, e.g., at least three Rp internucleoside linkages. The gap region may include, e.g., at least three Rp phosphorothioate internucleoside linkages. The gap region may include, e.g., at least 4, 5, 6, 7, 8, 9, or 10 Rp internucleoside linkages. The gap region may include, e.g., at least 4, 5, 6, 7, 8, 9, or 10 Rp phosphorothioate internucleoside linkages.

A gapmer may include a wing-gap-wing motif that is a 5-10-5 motif, where the nucleosides in each wing region are 2'-MOE-modified nucleosides. A wing-gap-wing motif of a gapmer may be, e.g., a 5-10-5 motif where the nucleosides in the gap region are 2'-deoxyribonucleosides. A wing-gap-wing motif of a gapmer may be, e.g., a 5-10-5 motif, where all internucleoside linkages are phosphorothioate internucleoside linkages. A wing-gap-wing motif of a gapmer may be, e.g., a 5-10-5 motif, where all internucleoside linkages are stereochemically enriched phosphorothioate internucleoside linkages. A wing-gap-wing motif of a gapmer may be, e.g., a 5-10-5 motif, where the nucleosides in each wing region are 2'-MOE-modified nucleosides, the nucleosides in the gap region are 2'-deoxyribonucleosides, and all internucleoside linkages are stereochemically enriched phosphorothioate internucleoside linkages.

In certain embodiments, a wing-gap-wing motif is a 5-10-5 motif where the residues at each wing region are not 2'-MOE-modified residues. In certain embodiments, a wing-gap-wing motif is a 5-10-5 motif where the residues in the gap region are 2'-deoxyribonucleotide residues. In certain embodiments, a wing-gap-wing motif is a 5-10-5 motif, where all internucleosidic linkages are phosphorothioate internucleosidic linkages. In certain embodiments, a wing-gap-wing motif is a 5-10-5 motif, where all internucleoside linkages are stereochemically enriched phosphorothioate internucleoside linkages. In certain embodiments, a wing- gap-wing motif is a 5-10-5 motif where the residues at each wing region are not 2'-MOE-modified residues, the residues in the gap region are 2'-deoxyribonucleotide, and all internucleoside linkages are stereochemically enriched phosphorothioate internucleoside linkages.

An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being a P-stereogenic linkage (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least two of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., stereogenic. At least three of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least four of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least five of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least six of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least seven of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least eight of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least nine of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P- stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). One of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Two of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Three of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Four of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P- stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Five of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Six of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Seven of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Eight of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P- stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Nine of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Ten of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages being P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least two of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P- stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least three of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least four of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least five of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least six of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least seven of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). One of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Two of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Three of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P- stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Four of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Five of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Six of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Seven of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Eight of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester).

An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester), and at least one internucleoside linkage being non-stereogenic. An oligonucleotide may include a region in which at least one of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages being P- stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester), and at least one internucleoside linkage being non-stereogenic. At least two internucleoside linkages may be, e.g., non-stereogenic. At least three internucleoside linkages may be, e.g., non-stereogenic. At least four internucleoside linkages may be, e.g., non-stereogenic. At least five internucleoside linkages may be, e.g., non-stereogenic. At least six internucleoside linkages may be, e.g., non-stereogenic. At least seven internucleoside linkages may be, e.g., non-stereogenic. At least eight internucleoside linkages may be, e.g., non-stereogenic. At least nine internucleoside linkages may be, e.g., non-stereogenic. At least 10 internucleoside linkages may be, e.g., non-stereogenic. At least 11 internucleoside linkages may be, e.g., non-stereogenic. At least 12 internucleoside linkages may be, e.g., non-stereogenic. At least 13 internucleoside linkages may be, e.g., non-stereogenic. At least 14 internucleoside linkages may be, e.g., non-stereogenic. At least 15 internucleoside linkages may be, e.g., non-stereogenic. At least 16 internucleoside linkages may be, e.g., non-stereogenic. At least 17 internucleoside linkages may be, e.g., non-stereogenic. At least 18 internucleoside linkages may be, e.g., non-stereogenic. At least 19 internucleoside linkages may be, e.g., non-stereogenic. At least 20 internucleoside linkages may be, e.g., non-stereogenic. In some embodiments, one internucleoside linkage is non-stereogenic. In some embodiments, two internucleoside linkages are non-stereogenic. In some embodiments, three internucleoside linkages are non-stereogenic. In some embodiments, four internucleoside linkages are non-stereogenic. In some embodiments, five internucleoside linkages are non-stereogenic. In some embodiments, six internucleoside linkages are non-stereogenic. In some embodiments, seven internucleoside linkages are non-stereogenic. In some embodiments, eight internucleoside linkages are non-stereogenic. In some embodiments, nine internucleoside linkages are non-stereogenic. In some embodiments, 10 internucleoside linkages are non-stereogenic. In some embodiments, 11 internucleoside linkages are non-stereogenic. In some embodiments, 12 internucleoside linkages are non-stereogenic. In some embodiments, 13 internucleoside linkages are non-stereogenic. In some embodiments, 14 internucleoside linkages are non-stereogenic. In some embodiments, 15 internucleoside linkages are non-stereogenic. In some embodiments, 16 internucleoside linkages are non-stereogenic. In some embodiments, 17 internucleoside linkages are non-stereogenic. In some embodiments, 18 internucleoside linkages are non-stereogenic. In some embodiments, 19 internucleoside linkages are non-stereogenic. In some embodiments, 20 internucleoside linkages are non-stereogenic. An oligonucleotide may include a region in which all internucleoside linkages, except at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages which is P-stereogenic, are non-stereogenic.

An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, and at least one internucleoside linkage being phosphate phosphodiester. An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, and at least one internucleoside linkage being phosphate phosphodiester. At least two internucleoside linkages may be, e.g., phosphate phosphodiesters. At least three internucleoside linkages may be, e.g., phosphate phosphodiesters. At least four internucleoside linkages may be, e.g., phosphate phosphodiesters. At least five internucleoside linkages may be, e.g., phosphate phosphodiesters. At least six internucleoside linkages may be, e.g., phosphate phosphodiesters. At least seven internucleoside linkages may be, e.g., phosphate phosphodiesters. At least eight internucleoside linkages may be, e.g., phosphate phosphodiesters. At least nine internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 10 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 11 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 12 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 13 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 14 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 15 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 16 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 17 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 18 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 19 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 20 internucleoside linkages may be, e.g., phosphate phosphodiesters. In some embodiments, one internucleoside linkage is phosphate phosphodiesters. In some embodiments, two internucleoside linkages are phosphate phosphodiesters.

In some embodiments, three internucleoside linkages are phosphate phosphodiesters. In some embodiments, four internucleoside linkages are phosphate phosphodiesters. In some embodiments, five internucleoside linkages are phosphate phosphodiesters. In some embodiments, six internucleoside linkages are phosphate phosphodiesters. In some embodiments, seven internucleoside linkages are phosphate phosphodiesters. In some embodiments, eight internucleoside linkages are phosphate phosphodiesters. In some embodiments, nine internucleoside linkages are phosphate phosphodiesters.

In some embodiments, 10 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 11 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 12 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 13 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 14 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 15 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 16 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 17 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 18 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 19 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 20 internucleoside linkages are phosphate phosphodiesters. An oligonucleotide may include a region with all internucleoside linkages, except at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, being phosphate phosphodiesters.

An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, and at least 10% of all internucleoside linkages in the region being non-stereogenic. An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, and at least 10% of all internucleoside linkages in the region being non-stereogenic. At least 20% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 30% of all the internucleoside linkages in the region may be, e.g., non- stereogenic. At least 40% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 50% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 60% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 70% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 80% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 90% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 50% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. A non-stereogenic internucleoside linkage may be, e.g., a phosphate phosphodiester. In some embodiments, each non-stereogenic internucleoside linkage is a phosphate phosphodiester. The first internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The first internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage. The second internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The second internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage. The third internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The third internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage. The fifth internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The fifth internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage. The seventh internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The seventh internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage. The eighth internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The eighth internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage. The ninth internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The ninth internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage. The eighteenth internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The eighteenth internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage. The nineteenth internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The nineteenth internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage. The twentieth internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The twentieth internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage.

The region may have a length of, e.g., at least 21 bases. The region may have a length of, e.g., 21 bases.

In some embodiments, each stereochemically enriched internucleoside linkage in an oligonucleotide is a phosphorothioate phosphodiester.

An oligonucleotide may have, e.g., at least 25% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 30% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 35% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 40% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 45% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 50% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 55% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 60% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 65% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 70% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 75% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 80% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 85% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 90% of its internucleoside linkages in Sp configuration.

An oligonucleotide may include at least two internucleoside linkages having different stereochemical configuration and/or different P-modifications relative to one another. The oligonucleotide may have a structure represented by the following formula:

[S^Bnl R^Bn2S^Bn3R^Bn4. . . S^BnxR^Bny] where: each R^B independently represents a block of nucleotide units having the Rp configuration at the internucleoside linkage phosphorus atom; each S^B independently represents a block of nucleotide units having the Sp configuration at the internucleoside linkage phosphorus atom; each of n1 to ny is zero or an integer, provided that at least one odd n and at least one even n must be non-zero so that the oligonucleotide includes at least two internucleoside linkages with different stereochemistry relative to one another; and where the sum of n1 to ny is between 2 and 200.

In some embodiments, the sum of n1 to ny is between a lower limit selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, and more, and the upper limit selected from the group consisting of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200, the upper limit being greater than the lower limit. In some of these embodiments, each n has the same value. In some embodiments, each even n has the same value as each other even n. In some embodiments, each odd n has the same value each other odd n. At least two even ns may have, e.g., different values from one another. At least two odd ns may have, e.g., different values from one another.

At least two adjacent ns may be, e.g., equal to one another, so that an oligonucleotide includes adjacent blocks of Sp linkages and Rp linkages of equal lengths. In some embodiments, an oligonucleotide includes repeating blocks of Sp and Rp linkages of equal lengths. In some embodiments, an oligonucleotide includes repeating blocks of Sp and Rp linkages, where at least two such blocks are of different lengths from one another. In some such embodiments, each Sp block is of the same length and is of a different length from each Rp block, where all Rp blocks may optionally be of the same length as one another.

At least two skip-adjacent ns may be, e.g., equal to one another, so that a provided oligonucleotide includes at least two blocks of internucleoside linkages of a first stereochemistry that are equal in length to one another and are separated by a separating block of internucleoside linkages of the opposite stereochemistry, where the separating block may be of the same length or a different length from the blocks of first stereochemistry.

In some embodiments, ns associated with linkage blocks at the ends of an oligonucleotide are of the same length. In some embodiments, an oligonucleotide has terminal blocks of the same linkage stereochemistry. In some such embodiments, the terminal blocks are separated from one another by a middle block of the opposite linkage stereochemistry.

An oligonucleotide of formula [S^Bni R^Bn2S^Bn3R^Bn4. . . S^BnxR^Bn_y] may be, e.g., a stereoblockmer.

An oligonucleotide of formula [S^Bni R^Bn2S^Bn3R^Bn4. . . S^BnxR^Bn_y] may be, e.g., a stereoskipmer. An oligonucleotide of formula [S^Bni R^Bn2S^Bn3R^Bn4. . . S^BnxR^Bn_y] may be, e.g., a stereoaltmer. An oligonucleotide of formula [S^Bni R^Bn2S^Bn3R^Bn4. . . S^BnxR^Bn_y] may be, e.g., a gapmer.

An oligonucleotide of formula [S^B _ni R^Bn2S^Bn3R^Bn4. . . S^BnxR^Bn_y] may be, e.g., of any of the above described patterns and may further include, e.g., patterns of P-modifications. For instance, an oligonucleotide of formula [S^Bni R^Bn2S^Bn3R^Bn4. . . S^BnxR^Bn_y] may be, e.g., a stereoskipmer and a P- modification skipmer. An oligonucleotide of formula [S^B _{n i} R^Bn2S^Bn3R^Bn4. . . S^BnxR^Bn_y] may be, e.g., a stereoblockmer and a P-modification altmer. An oligonucleotide of formula [S^B _niR^Bn₂S^Bn3R^Bn₄. . .

S^BnxR^Bn_y] may be, e.g., a stereoaltmer and a P-modification blockmer.

An oligonucleotide may include, e.g., at least one phosphate phosphodiester and at least two consecutive modified internucleoside linkages. An oligonucleotide may include, e.g., at least one phosphate phosphodiester and at least two consecutive phosphorothioate triesters.

An oligonucleotide may be, e.g., a blockmer. An oligonucleotide may be, e.g., a stereoblockmer. An oligonucleotide may be, e.g., a P-modification blockmer. An oligonucleotide may be, e.g., a linkage blockmer.

An oligonucleotide may be, e.g., an altmer. An oligonucleotide may be, e.g., a stereoaltmer. An oligonucleotide may be, e.g., a P-modification altmer. An oligonucleotide may be, e.g., a linkage altmer.

An oligonucleotide may be, e.g., a unimer. An oligonucleotide may be, e.g., a stereounimer. An oligonucleotide may be, e.g., a P-modification unimer. An oligonucleotide may be, e.g., a linkage unimer.

An oligonucleotide may be, e.g., a skipmer.

II. Complementarity

It is possible to introduce mismatch bases without eliminating activity. Accordingly an oligonucleotide of the invention may include (i) a nucleobase sequence having at least 6 contiguous nucleobases complementary to an equal-length portion within an NRL target nucleic acid and (ii) a nucleobase sequence having a plurality of nucleobases including one or more nucleobases complementary to an NRL target nucleic acid and one or more mismatches.

In some embodiments, oligonucleotides of the invention are complementary to an NRL target nucleic acid over the entire length of the oligonucleotide. In other embodiments, oligonucleotides are 99%, 95%, 90%, 85%, or 80% complementary to the NRL target nucleic acid. In further embodiments, oligonucleotides are at least 80% complementary to the NRL target nucleic acid over the entire length of the oligonucleotide and include a nucleobase sequence that is fully complementary to an NRL target nucleic acid. The nucleobase sequence that is fully complementary may be, e.g., 6 to 20, 10 to 18, or 18 to 20 contiguous nucleobases in length.

An oligonucleotide of the invention may include one or more mismatched nucleobases relative to the target nucleic acid. In certain embodiments, an antisense or RNAi activity against the target is reduced by such mismatch, but activity against a non-target is reduced by a greater amount. Thus, the off-target selectivity of the oligonucleotides may be improved.

III. Oligonucleotide Modifications

An oligonucleotide of the invention may be a modified oligonucleotide. A modified oligonucleotide of the invention includes one or more modifications, e.g., a nucleobase modification (e.g., SEQ ID NOs: 18-29), a sugar modification, an internucleoside linkage modification, or a terminal modification.

Nucleobase Modifications

Oligonucleotides of the invention may include one or more modified nucleobases. Unmodified nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-substituted pyrimidines, 6- azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6 substituted purines, as well as synthetic and natural nucleobases, e.g., 5-methylcytosine, 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl) adenine and guanine, 2-alkyl (e.g., 2-propyl) adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5- halouracil, 5-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 5-trifluoromethyl uracil, 5-trifluoromethyl cytosine, 7-methyl guanine, 7-methyl adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7- deazaadenine, 3-deazaguanine, 3-deazaadenine. Certain nucleobases are particularly useful for increasing the binding affinity of nucleic acids, e g., 5-substituted pyrimidines; 6-azapyrimidines; N2-, N6-, and/or 06-substituted purines. Nucleic acid duplex stability can be enhanced using, e.g., 5- methylcytosine. Non-limiting examples of nucleobases include: 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2- propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl ( — CºC — CH3) uracil, 5- propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8- halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5- bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F- adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N- benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N- benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1 ,3-diazaphenoxazine-2-one, 1 ,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-1 ,3- diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deazaadenine, 7-deazaguanine, 2- aminopyridine and 2-pyridone. Further nucleobases include those disclosed in Merigan et al., U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991 , 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press, 2008, 163-166 and 442-443

Sugar Modifications

Oligonucleotides of the invention may include one or more sugar modifications in nucleosides. Nucleosides having an unmodified sugar include a sugar moiety that is a furanose ring as found in ribonucleosides and 2’-deoxyribonucleosides.

Sugars included in the nucleosides of the invention may be non-furanose (or 4'-substituted furanose) rings or ring systems or open systems. Such structures include simple changes relative to the natural furanose ring (e.g., a six-membered ring). Alternative sugars may also include sugar surrogates wherein the furanose ring has been replaced with another ring system such as, e.g., a morpholino or hexitol ring system. Non-limiting examples of sugar moieties useful that may be included in the oligonucleotides of the invention include b-D-ribose, p-D-2'-deoxyribose, substituted sugars (e.g., 2', 5', and bis substituted sugars), 4'-S-sugars (e.g., 4'-S-ribose, 4'-S-2'-deoxyribose, and 4'-S-2'-substituted ribose), bridged sugars (e.g., the 2'-0 — CH₂-4' or 2'-0 — (CH₂)₂-4' bridged ribose derived bicyclic sugars) and sugar surrogates (when the ribose ring has been replaced with a morpholino or a hexitol ring system). The nucleosides and nucleotides containing bridged sugars having 2'-0 — CH₂-4' are referred to herein as locked nucleic acids (LNA).

Typically, a sugar modification may be, e.g., a 2’-substitution, locking, carbocyclization, or unlocking. A 2’-substitution is a replacement of 2’-hydroxyl in ribofuranose with 2’-fluoro, 2’-methoxy, or 2’-(2-methoxy)ethoxy. Alternatively, a 2’-substitution may be a 2’-(ara) substitution, which corresponds to the following structure:

where B is a nucleobase, and R is a 2’-(ara) substituent (e.g., fluoro). 2’-(ara) substituents are known in the art and can be same as other 2’-substituents described herein. In some embodiments, 2’-(ara) substituent is a 2’-(ara)-F substituent (R is fluoro). A locking modification is an incorporation of a bridge between 4’-carbon atom and 2’-carbon atom of ribofuranose. Nucleosides having a sugar with a locking modification are known in the art as bridged nucleic acids, e.g., locked nucleic acids (LNA), ethylene- bridged nucleic acids (ENA), and cEt nucleic acids. The bridged nucleic acids are typically used as affinity enhancing nucleosides.

Internucleoside Linkage Modifications

Oligonucleotides of the invention may include one or more internucleoside linkage modifications. The two main classes of internucleoside linkages are defined by the presence or absence of a phosphorus atom. Non-limiting examples of phosphorus-containing internucleoside linkages include phosphodiester linkages, phosphotriester linkages, phosphorothioate diester linkages, phosphorothioate triester linkages, morpholino internucleoside linkages, methylphosphonates, and phosphoramidate. Nonlimiting examples of non-phosphorus internucleoside linkages include methylenemethylimino ( — CH2 — N(CH3) — O — CH2 — ), thiodiester (— O— C(O)— S— ), thionocarbamate (— O— C(0)(NH)— S— ), siloxane (— O— Si(H)2— O— ), and N,N'-dimethylhydrazine ( — C H2 — N (C H 3) — N (C H 3) — ) . Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. Methods of preparation of phosphorous-containing and non- phosphorous-containing internucleoside linkages are known in the art.

Internucleoside linkages may be stereochemically enriched. For example, phosphorothioate- based internucleoside linkages (e.g., phosphorothioate diester or phosphorothioate triester) may be stereochemically enriched. The stereochemically enriched internucleoside linkages including a stereogenic phosphorus are typically designated Sp or Rp to identify the absolute stereochemistry of the phosphorus atom. Wthin an oligonucleotide, Sp phosphorothioate indicates the following structure:

Within an oligonucleotide, Rp phosphorothioate indicates the following structure:

The oligonucleotides of the invention may include one or more neutral internucleoside linkages. Non-limiting examples of neutral internucleoside linkages include phosphotriesters, phosphorothioate triesters, methylphosphonates, methylenemethylimino (3'-CH2 — N(CH3) — 0-3’), amide-3 (3'-CH2 —

C(=0) — N(H)-3’), amide-4 (3'-CH₂ — N(H) — C(=0)-3’), formacetal (3'-0— CH₂— 0-3’), and thioformacetal (3'-S — CH2 — 0-3’). Further neutral internucleoside linkages include nonionic linkages including siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester, and amides (See for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65).

Oligonucleotides may include, e.g., modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. Oligonucleotides may include, e.g., a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides of the present disclosure include a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide may include, e.g., a region that is uniformly linked by phosphorothioate internucleoside linkages. The oligonucleotide may be, e.g., uniformly linked by phosphorothioate internucleoside linkages. Each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. Each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.

The oligonucleotide may include, e.g., at least 6 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., at least 7 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., at least 8 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., at least 9 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., at least 10 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., at least 11 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., at least 12 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., at least 13 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., at least 14 phosphorothioate internucleoside linkages.

The oligonucleotide may include, e.g., at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., at least one block of at least 7 consecutive phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., at least one block of at least 9 consecutive phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., at least one block of at least 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3' end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3' end of the oligonucleotide. The oligonucleotide may include, e.g., fewer than 15 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 14 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 13 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 12 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 11 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 10 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 9 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 8 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 7 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 6 phosphorothioate internucleoside linkages. The oligonucleotide may include, e.g., fewer than 5 phosphorothioate internucleoside linkages. In some embodiments, at least one phosphorothioate internucleoside linkage is a phosphorothioate diester. In some embodiments, each phosphorothioate internucleoside linkage is a phosphorothioate diester. In some embodiments, at least one phosphorothioate internucleoside linkage is a phosphorothioate triester. In some embodiments, each phosphorothioate internucleoside linkage is a phosphorothioate triester. In some embodiments, each internucleoside linkage is independently a phosphodiester (e.g., phosphate phosphodiester or phosphorothioate diester).

An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (Sp)mRp or Rp(Sp)m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including Rp(Sp)_m. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (Sp)_mRp. In some embodiments, m is 2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including RP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (SP)2RP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (RP)2RP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including RPSPRP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including SPRPRP(SP)2. An oligonucleotide may include a pattern of internucleoside P-stereogenic centers including (SP)2RP.

In the embodiments of internucleoside P-stereogenic center patterns, m is 2, 3, 4, 5, 6, 7 or 8, unless specified otherwise. In some embodiments of internucleoside P-stereogenic center patterns, m is 3, 4, 5, 6, 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, m is 4, 5, 6, 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, m is 5, 6, 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, m is 6, 7 or 8. In some embodiments of internucleoside P-stereogenic center patterns, m is 7 or 8. In some embodiments of internucleoside P- stereogenic center patterns, m is 2. In some embodiments of internucleoside P-stereogenic center patterns, m is 3. In some embodiments of internucleoside P-stereogenic center patterns, m is 4. In some embodiments of internucleoside P-stereogenic center patterns, m is 5. In some embodiments of internucleoside P-stereogenic center patterns, m is 6. In some embodiments of internucleoside P- stereogenic center patterns, m is 7. In some embodiments of internucleoside P-stereogenic center patterns, m is 8.

An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being a P-stereogenic linkage (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least two of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., stereogenic. At least three of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least four of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least five of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least six of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least seven of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least eight of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least nine of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P- stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). One of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Two of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Three of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Four of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P- stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Five of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Six of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Seven of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Eight of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P- stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Nine of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Ten of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester).

An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages being P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least two of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P- stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least three of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least four of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least five of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least six of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). At least seven of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). One of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Two of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Three of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P- stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Four of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Five of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Six of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Seven of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester). Eight of the first, second, third, fifth, seventh, eighteenth, nineteenth and twentieth internucleoside linkages may be, e.g., P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester).

An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester), and at least one internucleoside linkage being non-stereogenic. An oligonucleotide may include a region in which at least one of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages being P- stereogenic (e.g., phosphorothioate phosphodiester or phosphorothioate phosphotriester), and at least one internucleoside linkage being non-stereogenic. At least two internucleoside linkages may be, e.g., non-stereogenic. At least three internucleoside linkages may be, e.g., non-stereogenic. At least four internucleoside linkages may be, e.g., non-stereogenic. At least five internucleoside linkages may be, e.g., non-stereogenic. At least six internucleoside linkages may be, e.g., non-stereogenic. At least seven internucleoside linkages may be, e.g., non-stereogenic. At least eight internucleoside linkages may be, e.g., non-stereogenic. At least nine internucleoside linkages may be, e.g., non-stereogenic. At least 10 internucleoside linkages may be, e.g., non-stereogenic. At least 11 internucleoside linkages may be, e.g., non-stereogenic. At least 12 internucleoside linkages may be, e.g., non-stereogenic. At least 13 internucleoside linkages may be, e.g., non-stereogenic. At least 14 internucleoside linkages may be, e.g., non-stereogenic. At least 15 internucleoside linkages may be, e.g., non-stereogenic. At least 16 internucleoside linkages may be, e.g., non-stereogenic. At least 17 internucleoside linkages may be, e.g., non-stereogenic. At least 18 internucleoside linkages may be, e.g., non-stereogenic. At least 19 internucleoside linkages may be, e.g., non-stereogenic. At least 20 internucleoside linkages may be, e.g., non-stereogenic. In some embodiments, one internucleoside linkage is non-stereogenic. In some embodiments, two internucleoside linkages are non-stereogenic. In some embodiments, three internucleoside linkages are non-stereogenic. In some embodiments, four internucleoside linkages are non-stereogenic. In some embodiments, five internucleoside linkages are non-stereogenic. In some embodiments, six internucleoside linkages are non-stereogenic. In some embodiments, seven internucleoside linkages are non-stereogenic. In some embodiments, eight internucleoside linkages are non-stereogenic. In some embodiments, nine internucleoside linkages are non-stereogenic. In some embodiments, 10 internucleoside linkages are non-stereogenic. In some embodiments, 11 internucleoside linkages are non-stereogenic. In some embodiments, 12 internucleoside linkages are non-stereogenic. In some embodiments, 13 internucleoside linkages are non-stereogenic. In some embodiments, 14 internucleoside linkages are non-stereogenic. In some embodiments, 15 internucleoside linkages are non-stereogenic. In some embodiments, 16 internucleoside linkages are non-stereogenic. In some embodiments, 17 internucleoside linkages are non-stereogenic. In some embodiments, 18 internucleoside linkages are non-stereogenic. In some embodiments, 19 internucleoside linkages are non-stereogenic. In some embodiments, 20 internucleoside linkages are non-stereogenic. An oligonucleotide may include a region in which all internucleoside linkages, except at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleoside linkages which is P-stereogenic, are non-stereogenic.

An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, and at least one internucleoside linkage being phosphate phosphodiester. An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, and at least one internucleoside linkage being phosphate phosphodiester. At least two internucleoside linkages may be, e.g., phosphate phosphodiesters. At least three internucleoside linkages may be, e.g., phosphate phosphodiesters. At least four internucleoside linkages may be, e.g., phosphate phosphodiesters. At least five internucleoside linkages may be, e.g., phosphate phosphodiesters. At least six internucleoside linkages may be, e.g., phosphate phosphodiesters. At least seven internucleoside linkages may be, e.g., phosphate phosphodiesters. At least eight internucleoside linkages may be, e.g., phosphate phosphodiesters. At least nine internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 10 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 11 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 12 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 13 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 14 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 15 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 16 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 17 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 18 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 19 internucleoside linkages may be, e.g., phosphate phosphodiesters. At least 20 internucleoside linkages may be, e.g., phosphate phosphodiesters. In some embodiments, one internucleoside linkage is phosphate phosphodiesters. In some embodiments, two internucleoside linkages are phosphate phosphodiesters.

In some embodiments, three internucleoside linkages are phosphate phosphodiesters. In some embodiments, four internucleoside linkages are phosphate phosphodiesters. In some embodiments, five internucleoside linkages are phosphate phosphodiesters. In some embodiments, six internucleoside linkages are phosphate phosphodiesters. In some embodiments, seven internucleoside linkages are phosphate phosphodiesters. In some embodiments, eight internucleoside linkages are phosphate phosphodiesters. In some embodiments, nine internucleoside linkages are phosphate phosphodiesters. In some embodiments, 10 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 11 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 12 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 13 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 14 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 15 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 16 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 17 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 18 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 19 internucleoside linkages are phosphate phosphodiesters. In some embodiments, 20 internucleoside linkages are phosphate phosphodiesters. An oligonucleotide may include a region with all internucleoside linkages, except at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, being phosphate phosphodiesters.

An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, and at least 10% of all internucleoside linkages in the region being non-stereogenic. An oligonucleotide may include a region with at least one of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleoside linkages being P-stereogenic, and at least 10% of all internucleoside linkages in the region being non-stereogenic. At least 20% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 30% of all the internucleoside linkages in the region may be, e.g., non- stereogenic. At least 40% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 50% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 60% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 70% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 80% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 90% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. At least 50% of all the internucleoside linkages in the region may be, e.g., non-stereogenic. A non-stereogenic internucleoside linkage may be, e.g., a phosphate phosphodiester. In some embodiments, each non-stereogenic internucleoside linkage is a phosphate phosphodiester.

The first internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The first internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage. The second internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The second internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage. The third internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The third internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage. The fifth internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The fifth internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage. The seventh internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The seventh internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage. The eighth internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The eighth internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage. The ninth internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The ninth internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage. The eighteenth internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The eighteenth internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage. The nineteenth internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The nineteenth internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage. The twentieth internucleoside linkage of the region may be, e.g., an Sp internucleoside linkage. The twentieth internucleoside linkage of the region may be, e.g., an Rp internucleoside linkage.

The region may have a length of, e.g., at least 21 bases. The region may have a length of, e.g., 21 bases.

In some embodiments, each stereochemically enriched internucleoside linkage in an oligonucleotide is a phosphorothioate phosphodiester.

An oligonucleotide may have, e.g., at least 25% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 30% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 35% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 40% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 45% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 50% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 55% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 60% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 65% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 70% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 75% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 80% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 85% of its internucleoside linkages in Sp configuration. An oligonucleotide may have, e.g., at least 90% of its internucleoside linkages in Sp configuration.

An oligonucleotide may include, e.g., at least one phosphate phosphodiester and at least two consecutive modified internucleoside linkages. An oligonucleotide may include, e.g., at least one phosphate phosphodiester and at least two consecutive phosphorothioate triesters.

An oligonucleotide may be, e.g., a blockmer. An oligonucleotide may be, e.g., a stereoblockmer. An oligonucleotide may be, e.g., a P-modification blockmer. An oligonucleotide may be, e.g., a linkage blockmer.

An oligonucleotide may be, e.g., an altmer. An oligonucleotide may be, e.g., a stereoaltmer. An oligonucleotide may be, e.g., a P-modification altmer. An oligonucleotide may be, e.g., a linkage altmer.

An oligonucleotide may be, e.g., a unimer. An oligonucleotide may be, e.g., a stereounimer. An oligonucleotide may be, e.g., a P-modification unimer. An oligonucleotide may be, e.g., a linkage unimer. An oligonucleotide may be, e.g., a skipmer.

Terminal Modifications

Oligonucleotides of the invention may include a terminal modification. The terminal modification is a 5’-terminal modification or a 3’-terminal modification.

The 5’ end of an oligonucleotide may be, e.g., hydroxyl, a hydrophobic moiety, 5’ cap, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, diphosphrodithioate, triphosphorodithioate, phosphonate, phosphoramidate, a cell penetrating peptide, an endosomal escape moiety, or a neutral organic polymer. An unmodified 5’- terminus is hydroxyl or phosphate. An oligonucleotide having a 5’ terminus other than 5’-hydroxyl or 5’- phosphate has a modified 5’ terminus.

The 3’ end of an oligonucleotide may be, e.g., hydroxyl, a hydrophobic moiety, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, disphorodithioate, triphosphorodithioate, phosphonate, phosphoramidate, a cell penetrating peptide, an endosomal escape moiety, or a neutral organic polymer (e.g., polyethylene glycol). An unmodified 3’-terminuns is hydroxyl or phosphate. An oligonucleotide having a 3’ terminus other than 3’-hydroxyl or 3’-phosphate has a modified 3’ terminus.

The terminal modification (e.g., 5’-terminal modification) may be, e.g., a hydrophobic moiety. Advantageously, an oligonucleotide including a hydrophobic moiety may exhibit superior cellular uptake, as compared to an oligonucleotide lacking the hydrophobic moiety. Oligonucleotides including a hydrophobic moiety may therefore be used in compositions that are substantially free of transfecting agents. A hydrophobic moiety is a monovalent group (e.g., a bile acid (e.g., cholic acid, taurocholic acid, deoxycholic acid, oleyl lithocholic acid, or oleoyl cholenic acid), glycolipid, phospholipid, sphingolipid, isoprenoid, vitamin, saturated fatty acid, unsaturated fatty acid, fatty acid ester, triglyceride, pyrene, porphyrine, texaphyrine, adamantine, acridine, biotin, coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin, dimethoxytrityl, f-butydimethylsilyl, f-butyldiphenylsilyl, cyanine dye (e.g., Cy3 or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen) covalently linked to the terminus of the oligonucleotide backbone (e.g., 5’-terminus). Non-limiting examples of the monovalent group include ergosterol, stigmasterol, b-sitosterol, campesterol, fucosterol, saringosterol, avenasterol, coprostanol, cholesterol, vitamin A, vitamin D, vitamin E, cardiolipin, and carotenoids. The linker connecting the monovalent group to the oligonucleotide may be an optionally substituted Ci-eo aliphatic (e.g., optionally substituted Ci-eo alkylene) or an optionally substituted C2-60 heteroaliphatic (e.g., optionally substituted C2-60 heteroalkylene), where the linker may be optionally interrupted with one, two, or three instances independently selected from the group consisting of an optionally substituted arylene, optionally substituted heterocyclylene, and optionally substituted cycloalkylene. The linker may be bonded to an oligonucleotide through, e.g., an oxygen atom attached to a 5’-terminal carbon atom, a 3’-terminal carbon atom, a 5’-terminal phosphate or phosphorothioate, a 3’-terminal phosphate or phosphorothioate, or an internucleoside linkage.

IV. Pharmaceutical Compositions

An oligonucleotide of the invention (e.g., a pharmaceutically acceptable salt thereof) may be included in a pharmaceutical composition. A pharmaceutical composition typically includes a pharmaceutically acceptable diluent or carrier. A pharmaceutical composition may include (e.g., consist of), e.g., a sterile saline solution and an oligonucleotide of the invention. The sterile saline is typically a pharmaceutical grade saline. A pharmaceutical composition may include (e.g., consist of), e.g., sterile water and an oligonucleotide of the invention. The sterile water is typically a pharmaceutical grade water. A pharmaceutical composition may include (e.g., consist of), e.g., phosphate-buffered saline (PBS) and an oligonucleotide of the invention. The sterile PBS is typically a pharmaceutical grade PBS.

In certain embodiments, pharmaceutical compositions include one or more oligonucleotides and one or more excipients. In certain embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, oligonucleotides may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

In certain embodiments, pharmaceutical compositions including an oligonucleotide encompass any pharmaceutically acceptable salts of the oligonucleotide, esters of the oligonucleotide, or salts of such esters. In certain embodiments, pharmaceutical compositions including an oligonucleotide, upon administration to a subject (e.g., a human), are capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of oligonucleotides, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. In certain embodiments, prodrugs include one or more conjugate group attached to an oligonucleotide, wherein the conjugate group is cleaved by endogenous nucleases within the body.

Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid, such as an oligonucleotide, is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.

In certain embodiments, pharmaceutical compositions include a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those including hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.

In certain embodiments, pharmaceutical compositions include one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.

In certain embodiments, pharmaceutical compositions include a co-solvent system. Certain of such co-solvent systems include, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol including 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

In certain embodiments, pharmaceutical compositions are prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration. In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intraocular (e.g., intravitreal), intravenous, subcutaneous, intramuscular, intrathecal, intracerebroventricular, etc.). In certain of such embodiments, a pharmaceutical composition includes a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multidose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain.

V. Methods of the Invention

The invention provides methods of using oligonucleotides (e.g., pharmaceutically salts thereof) and pharmaceutical compositions of the invention.

A method of the invention may be a method of inhibiting or reducing the production of an NRL protein in a cell including an NRL gene by contacting the cell with the oligonucleotide (e.g., a pharmaceutically salt thereof) of the invention. The cell may be present in a subject (e.g., in a subject’s eye). The cell may be a photoreceptor cell.

A method of the invention may be a method of treating a subject having a disease, disorder, or condition (e.g., retinitis pigmentosa) by administering to the subject a therapeutically effective amount of an oligonucleotide of the invention or a pharmaceutical composition of the invention. The diseases, disorders, and conditions that may be treated using methods of the invention include retinitis pigmentosa (e.g., Rho P23H-associated retinitis pigmentosa, PDE6-associated retinitis pigmentosa, MERTK- associated retinitis pigmentosa, BBS1 -associated retinitis pigmentosa, Rho-associated retinitis pigmentosa, MRFP-associated retinitis pigmentosa, RLBP1 -associated retinitis pigmentosa, RP1- associated retinitis pigmentosa, RPGR-X-linked retinitis pigmentosa, NR2E3-associated retinitis pigmentosa, or SPATA7-associated retinitis pigmentosa), Stargardt disease (e.g., ABCA4-associated Stargardt disease), cone-rod dystrophy (e.g., AIPL1 -associated cone-rod dystrophy or RGRIP1- associated cone-rod dystrophy), Leber congenital amaurosis (e.g., AIPL1 -associated Leber congenital amaurosis, GUCY2D-associated Leber congenital amaurosis, RD3-associated Leber congenital amaurosis, RPE65-associated Leber congenital amaurosis, or SPATA7-associated Leber congenital amaurosis), Bardet Biedl syndrome (e.g., BBS1 -associated Bardet Biedl syndrome), macular dystrophy (e.g., BEST1 -associated macular dystrophy), dry macular degeneration, geographic atrophy, atrophic age-related macular degeneration (AMD), advanced dry AMD, retinal dystrophy (e.g., CEP290- associated retinal dystrophy, CDH3-associated retinal dystrophy, CRB1 -associated retinal dystrophy, or PRPH2-associated retinal dystrophy), choroideremia (e.g., CHM-associated choroideremia), Usher syndrome type 1 (e.g., MY07A-associated Usher syndrome), retinoschisis (e.g., RS1-X-linked retinoschisis), Leber hereditary optic neuropathy (e.g., ND4-associated Lebe’rs hereditary optic neuropathy), and achromatopsia (e.g., CNGA3-associated achromatopsia or CNGB3-associated achromatopsia). Methods of the invention may be used to treat subjects having a disease, disorder, or condition associated with a dysfunction of ABCA4, AIPL1 , BBS1 , BEST1 , CEP290, CDH3, CHM, CNGA3, CNGB3, CRB1 , GUCY2D, MERTK, MRFP, MY07A, ND4, NR2E3, PDE6, PRPH2, RD3, RHO, RLBP1 , RP1 , RPE65, RPGR, RPGRIP1 , RS1 , or SPAT A7 gene. Advantageously, because the oligonucleotides of the invention target NR2E3 and not ABCA4, AIPL1 , BBS1 , BEST1 , CEP290, CDH3, CHM, CNGA3, CNGB3, CRB1 , GUCY2D, MERTK, MRFP, MY07A, ND4, PDE6, PRPH2, RD3, RHO, RLBP1 , RP1 , RPE65, RPGR, RPGRIP1 , RS1 , or SPATA7, the therapeutic activity of the oligonucleotides of the invention does not depend on the type of the mutation responsible for the dysfunctional ABCA4, AIPL1 , BBS1 , BEST1 , CEP290, CDH3, CHM, CNGA3, CNGB3, CRB1 , GUCY2D, MERTK, MRFP, MY07A,

ND4, NR2E3, PDE6, PRPH2, RD3, RHO, RLBP1 , RP1 , RPE65, RPGR, RPGRIP1 , RS1 , or SPATA7 gene.

The oligonucleotide of the invention (e.g., a pharmaceutically salt thereof) or the pharmaceutical composition of the invention may be administered to the subject using methods known in the art. For example, the oligonucleotide of the invention (e.g., a pharmaceutically salt thereof) or the pharmaceutical composition of the invention may be administered topically to the eye of the subject. Additionally or alternatively, the oligonucleotide (e.g., a pharmaceutically salt thereof) of the invention or the pharmaceutical composition of the invention may be administered to the subject intraocularly (e.g., intravitreally).

VI. Preparation of Oligonucleotides

Oligonucleotides of the invention may be prepared using techniques and methods known in the art for the oligonucleotide synthesis. For example, oligonucleotides of the invention may be prepared using a phosphoramidite-based synthesis cycle. This synthesis cycle includes the steps of (1) deblocking a 5’-protected nucleotide to produce a 5’-deblocked nucleotide, (2) coupling the 5’-deblocked nucleotide with a 5’-protected nucleoside phosphoramidite to produce nucleosides linked through a phosphite, (3) repeating steps (1) and (2) one or more times as needed, (4) capping the 5’-terminus, and (5) oxidation orsulfurization of internucleoside phosphites. The reagents and reaction conditions useful for the oligonucleotide synthesis are known in the art.

The oligonucleotides disclosed herein may be linked to solid support as a result of solid-phase synthesis. Cleavable solid supports that may be used with the oligonucleotides are known in the art. Non-limiting examples of the solid support include, e.g., controlled pore glass or macroporous polystyrene bonded to a strand through a cleavable linker (e.g., succinate-based linker) known in the art (e.g., UnyLinker™). An oligonucleotide linked to solid support may be removed from the solid support by cleaving the linker connecting an oligonucleotide and solid support. The following examples are meant to illustrate the invention. They are not meant to limit the invention in any way.

EXAMPLES

Example 1. Preparation of Single-Stranded Oligonucleotides Oligonucleotides may be prepared using techniques and methods known in the art. Single- stranded oligonucleotides of SEQ ID NOs: 6-17 may be, e.g., gapmers with all internucleoside linkages being phosphorothioate diesters. The antisense oligonucleotides (SEQ ID NOs: 6-17) and modified versions thereof (SEQ ID NOs: 18-29) are listed in Table 1.

Table 1: Nrl-targeting oligonucleotides

Chemical modifications above include the following: Each “+” refers to an LNA nucleotide, such that +A refers to an LNA nucleotide having adenine nucleobase. Each “*” refers to a phosphorothioate internucleoside linkage. Each “iMe-dC” refers to 5-methyl-dC nucleotide. All nucleosides in Table 1 are deoxyribonucleosides, unless otherwise noted (e.g., LNA).

Example 2. Inhibition of Target Nucleic Acid Expression In Vitro

Oligonucleotides were assessed for their ability to knockdown a target NRL nucleic acid in cultured HEK293 cells expressing high levels of the target homo sapiens NRL (GenBank ID:

BC012395.1) or macaca fascicularis NRL (GenBank ID: XM_015453620.1). Select oligonucleotides from Table 1 above (SEQ ID NOs: 19 and 21-29) were incubated with the cultured cells expressing high levels of the target NRL nucleic acid. Relative target NRL protein (the product of the NRL nucleic acid) reduction was determined using standard techniques useful for quantification of protein (Western blots). The measured target NRL protein was normalized to housekeeping gene (actin) or a constitutively expressed marker (GFP). For comparison, the measured target NRL protein levels was normalized to target NRL protein levels in the same cell line treated with a vehicle control (DPBS).

Dose response analysis was conducted in the selected cell lines (FIG. 2 and FIG. 3), demonstrating that the Nrl-targeting oligonucleotides are effective at reducing the expression of the target NRL. From the dose response analysis, the half maximal inhibitory concentration (IC50) was calculated. The IC50 values of all tested oligonucleotides, normalized to actin or GFP, in HEK293 cells expressing Homo sapiens and/or Macaca fascicularis NRL are indicated in Table 2 below. The relative nucleotide positions and regions for oligonucleotide binding to NRL are also shown below in Table 2.

Table 2: Relative location and IC50 of Nrl-targeting oligonucleotides

Example 3. Inhibition of Target Nucleic Acid Expression In Vitro

Oligonucleotides may be assessed for their ability to knockdown a target NRL nucleic acid in cultured cell line expressing high levels of the target NRL nucleic acid. Selected oligonucleotides may be incubated with a cultured cell line expressing high levels of the target NRL nucleic acid. Relative target NRL protein (a product of the NRL nucleic acid) reduction may be determined using standard techniques useful for quantification of proteins. For comparison, the measured target NRL nucleic acid levels may be normalized to target NRL protein levels in a cell treated with a vehicle control. Alternatively, the measured target NRL nucleic acid levels may be normalized to housekeeping gene levels.

A positive control oligonucleotide may be transfected to ensure appropriate cell transfection efficacy. The transfection may be effected using a transfection agent, e.g., LIPOFECTAMINE.

Dose response analysis may be conducted in the selected cell line. Dose-responsive reduction in the target NRL nucleic acid levels indicates that an oligonucleotide is effective at reducing the expression of the target NRL nucleic acid. Example 4. Functional Testing of Oligonucleotides in Explanted Retinal Cells

An oligonucleotide may be tested in a physiologically relevant primary culture assay using, e.g., intact retinas from wt mice. In this assay, suppression of Rho expression may be used as a read out. After a culture period with media containing vehicle or an oligonucleotide, the retinas may be collected and assessed for Rho expression. Rho is a well-described target of NRL in rod photoreceptors. Oligonucleotides of the invention may cause a substantial reduction in the Rho expression compared to a vehicle in retinal explants from mice. NRL loss-of-function mutations typically lead to a reduction in rod gene expression. To determine whether the same was true for our oligos, explant cultures of murine retinas treated as described above may also be assayed for rod photoreceptor genes, e.g., NRL, NR2E3, GNAT1 , PDE6A, PDE6B, RHO, GNB1 , and CRX. After a culture period (e.g., after 2, 3, 4, 5, 6, or 7 days), an oligonucleotide may decrease the expression of the rod specific genes compared to vehicle treatment.

Intact retinas from adult wild type C57BL/6J mice were explanted on 0.4 pm pore tissue culture inserts (Millicell) in media (Neurobasal-A containing 1% FBS, 1% N2, 1% B27, 1% Pen/Strep, and 0.5% L-Glutamine) containing scrambled negative control (AACACGTCTATACGC) or Nrl targeting (CACGATGCTCAGAAGT) antisense oligonucleotide (ASO) at 10 or 20 pM. In the sequences of the negative control and the ASO, bold, italicized, capital letters designate LNA nucleotides, and plain capital letters designed deoxyribonucleotides. In the sequences of the negative control and the ASO, all internucleoside linkages were phosphorothioate diesters. After 48h in vitro, RNA from the retinas was isolated using Qiazol (Qiagen) and cDNA was synthesized using the iScript cDNA synthesis kit (Bio- Rad). SYBR green PCR master mix (Applied Biosystems) was used for quantitative real-time PCR using a StepOnePlus real-time PCR system (Applied Biosystems). For analysis, values were normalized to Gapdh (DC_T) and DDC_T between control and ASO-treated samples was expressed as percent of controls (100*2^ADDCt). The following primer sequences were used (in order set forth as SEQ ID NOs: 30-49):

Gapdh (F: GGCATT GCT CT CAAT G ACAA, R: CTTGCTCAGT GTCCTT GCT G) ,

Nrl (F: TCCCAGTCCCTTGGCTATGG, R: CACCGAGCTGTATGGTGTG),

Nr2e3 (F: GCAGTGGATCCCACAGAGTT, R: GAGCAATTTCCCAAACCTCA)

Rhodopsin (F: CCCTTCTCCAACGTCACAGG, R: TGAGGAAGTTGATGGGGAAGC),

Gnatl (F: TGACCAATAATGAAGCCATGCT, R: TCTTTGCCCATGGTTGATCA),

S Opsin (F: CAGCATCCGCTTCAACTCCAA, R: GCAGATGAGGGAAAGAGGAATGA),

M Opsin (F: TTGCTGACCTAGCAGAGACCA, R: AGCCTTCAAT G AC AC AC AG AG) ,

Gnat2 (F: TCCACCTCAGTATCTGTTTTCC, R: TT GAG GT C AAGG AACT G ACT CTT) ,

Gnb3 (F: CACTGGCCATGAGTCAGACA, R: GCAGGAGGCGTCATCTGAG), and Otx2 (F: CCGCCTTACGCAGTCAATG, R: GAGGGATGCAGCAAGTCCATA).

The results of this study are shown in FIG. 1. The data summarized in FIG. 1 shows that treatment with the Nrl-targeting ASO decreased Nrl and rod gene expression without decreasing cone gene expression or the expression of Otx2. Otx2 is an upstream transcription factor expressed in both rod and cone photoreceptors. Example 5. Rhodopsin Expression Reduction in the Retinas of Adult Mice

Oligonucleotides may be tested for their effect on adult photoreceptor gene expression in vivo. Oligonucleotide compositions may be administered intravitreally to one eye of an adult mouse (>P21). After a predetermined period of time (e.g., 1 week, 1 month, 3 months, and/or 6 months following the administration), expression of photoreceptor genes may be measured in the treated eye and in the untreated eye. The photoreceptor gene expressions in the treated eye may then be compared to those in the untreated eye. Treatment with oligonucleotides of the invention may reduce the expression of Rho and rod specific genes, e.g., NRL, NR2E3, GNAT1 , PDE6A, PDE6B, GNB1 , and CRX. The Rho and the other rod specific gene expressions may be assessed by qPCR (nucleic acid) and by Western blot (proteins) analyses. The oligonucleotides may also increase the expression of some cone photoreceptor genes (e.g., GNAT 2, PDE6C, GNB3, OPN 1 SW, OPN 1 MW, ARR3, and/or THRB) in the adult retinas.

Example 6. Rod Degeneration in Mutant Rhodopsin Retinas

The effect of the oligonucleotides of the invention on Rho expression in adult rods may have potential as a way to slow the degeneration of these cells in dominant forms of retinitis pigmentosa, e.g., Rho P23H. In this disease, the affected individuals express a mutant form of rhodopsin that is likely inappropriately processed and ultimately leads to the death of the rods. Reducing the NRL expression using oligonucleotides of the invention may slow the degeneration of the rods.

The assay for assessing the effect of an oligonucleotide of the invention on retinitis pigmentosa may be performed as follows. Retina from RhoP23H transgenic mice at P8 may be explanted and maintained in media containing vehicle or an oligonucleotide of the invention. The majority of rod cell deaths in the RhoP23H transgenic line typically occurs between P14 and P21 . Therefore, explants of retinas from RhoP23H mice at P12 were made and treated the explants with vehicle or an oligonucleotide of the invention. Here, designations P8, P12, P14, and P21 refer to the post-natal age of the test mice.

In these tests, P8 explants allow for the assessment of the activity of the oligonucleotides of the invention in decreasing the level of expression of Rho, and P12 explants allow for the assessment of the activity of the oligonucleotides of the invention in preserving the cells in the outer nuclear layer (ONL).

After an extended culture period, the retinas may be subjected to histologic analysis. The number of nuclei may be counted in the outer nuclear layer (ONL) of each retina in the central region. Retinas treated with an oligonucleotide of the invention may have a greater number of rod photoreceptors in the ONL than vehicle-treated controls.

Example 7. Rod Degeneration in Mutant RPE Retinas

Oligonucleotides of the invention may slow the degeneration of adult rod cells in recessive forms of retinitis pigmentosa, driven by mutations in genes like Phosphodiesterase 6 (PDE6). PDE6 is highly concentrated in the retina. It is most abundant in the internal membranes of retinal photoreceptors, where it reduces cytoplasmic levels of cyclic guanosine monophosphate (cGMP) in rod and cone outer segments in response to light. In this disease, the affected individuals express a mutant form of PDE6 that ultimately leads to the death of the rods and cones.

Oligonucleotides of the invention may be assayed to assess their effect on the degeneration of the photoreceptor cells as follows. Retina from rd10 mice, carrying a spontaneous PDE mutation, at P8 may be explanted and maintained in media containing vehicle or an oligonucleotide of the invention. The mutant rods may then be assayed for the rhodopsin expression levels, and the rhodopsin expression levels may be compared to those in the wild-type retina. Rod degeneration in these mice starts around P18. Therefore, explants of retinas from rd10 mice at P16 may be made. The explants may be treated with vehicle or an oligonucleotide of the invention. Here, designations P8, P16, and P18 refer to the postnatal age of the test mice. In these tests, P8 explants allow for the assessment of the activity of the oligonucleotides of the invention in decreasing the level of expression of RHO, and P16 explants allow for the assessment of the activity of the oligonucleotides of the invention in preserving the cells in the outer nuclear layer (ONL).

After an extended culture period, the retinas may be subjected to histologic analysis. The number of nuclei may be counted in the ONL of each retina in the central region. Retinas treated with an oligonucleotide of the invention may have a greater number of rod photoreceptors in the ONL than vehicle-treated controls.

The studies described herein demonstrate that the oligonucleotides of the invention may be useful in the treatment of multiple inherited retinal degenerations (IRDs) in a mutation independent manner. The inherited retinal degenerations include, e.g., diseases, disorders, and conditions associated with a of ABCA4, AIPL1 , BBS1 , BEST1 , CEP290, CDH3, CHM, CNGA3, CNGB3, CRB1 , GUCY2D, MERTK, MRFP, MY07A, ND4, NRL, PDE6, PRPH2, RD3, RHO, RLBP1 , RP1 , RPE65, RPGR,

RPGRIP1 , RS1 , or SPATA7 gene. Non-limiting examples of the diseases, disorders, and conditions that may be treated using oligonucleotides of the invention include retinitis pigmentosa, Stargardt disease, cone-rod dystrophy, Leber congenital amaurosis, Bardet Biedl syndrome, macular dystrophy, dry macular degeneration, geographic atrophy, atrophic age-related macular degeneration (AMD), advanced dry AMD, retinal dystrophy, choroideremia, Usher syndrome type 1 , retinoschisis, Leber hereditary optic neuropathy, and achromatopsia.

Example 8. Primary Culture Assay for Assessing Photoreceptor Gene Expression in Human Retinas

An oligonucleotide may be tested in a physiologically relevant primary culture assay using intact human retinas. In this assay, suppression of NRL and additional rod genes (e.g., NR2E3, RHO, GNAT1 , PDE6A, PDE6B, GNB1 , and/or CRX) may be used as a read out. After a culture period (e.g., 2, 3, 4, 5,

6, or 7 days) with media containing vehicle or an oligonucleotide, the retinal tissue may be collected and assessed for rod gene expression. NR2E3, RHO, GNAT1 , PDE6A, PDE6B, and GNB1 are targets of NRL in photoreceptors, and oligonucleotides described herein may cause a substantial reduction of their expression. NRL loss-of-function mutations lead to a reduction in rod gene expression. To determine whether the same is true for oligonucleotides described herein, cultures of human retinas may be explanted and treated as described in Example 4.

Some embodiments of the invention are within the description provided above. Some embodiments are within the description of the following numbered paragraphs.

1. A single-stranded oligonucleotide comprising a total of 12 to 50 interlinked nucleotides and having a nucleobase sequence comprising at least 6 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof. 2. A single-stranded oligonucleotide comprising a total of 12 to 50 interlinked nucleotides and having a nucleobase sequence comprising at least 6 contiguous nucleobases complementary to the target sequence of any one of SEQ ID NOs: 6-17.

3. The oligonucleotide of paragraph 1 or 2, wherein the oligonucleotide comprises at least one modified nucleobase.

4. The oligonucleotide of paragraph 3, wherein at least one modified nucleobase is 5- methylcytosine.

5. The oligonucleotide of paragraph 3 or 4, wherein at least one modified nucleobase is 7- deazaguanine.

6. The oligonucleotide of any one of paragraphs 3 to 5, wherein at least one modified nucleobase is 6-thioguanine.

7. The oligonucleotide of any one of paragraphs 1 to 6, wherein the oligonucleotide comprises at least one modified internucleoside linkage.

8. The oligonucleotide of paragraph 7, wherein the modified internucleoside linkage is a phosphorothioate linkage.

9. The oligonucleotide of paragraph 8, wherein the phosphorothioate linkage is a stereochemically enriched phosphorothioate linkage.

10. The oligonucleotide of any one of paragraphs 7 to 9, wherein at least 50% of internucleoside linkages in the oligonucleotide are each independently the modified internucleoside linkage.

11. The oligonucleotide of paragraph 10, wherein at least 70% of internucleoside linkages in the oligonucleotide are each independently the modified internucleoside linkage.

12. The oligonucleotide of any one of paragraphs 1 to 11 , wherein the oligonucleotide comprises at least one modified sugar nucleoside.

13. The oligonucleotide of paragraph 12, wherein at least one modified sugar nucleoside is a bridged nucleic acid.

14. The oligonucleotide of paragraph 13, wherein the bridged nucleic acid is a locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), or cEt nucleic acid.

15. The oligonucleotide of paragraph 14, wherein the oligonucleotide is a gapmer.

16. The oligonucleotide of any one of paragraphs 12 to 15, wherein at least one modified sugar nucleoside is a 2’-modified sugar nucleoside.

17. The oligonucleotide of paragraph 16, wherein at least one 2’-modified sugar nucleoside comprises a 2’-modification selected from the group consisting of 2’-fluoro, 2’-methoxy, and 2’- methoxyethoxy.

18. The oligonucleotide of any one of paragraphs 1 to 17, wherein the oligonucleotide comprises deoxy ribonucleotides.

19. The oligonucleotide of any one of paragraphs 1 to 18, wherein the oligonucleotide comprises ribonucleotides.

20. The oligonucleotide of any one of paragraphs 1 to 6, wherein the oligonucleotide is a morpholino oligomer.

21. The oligonucleotide of any one of paragraphs 1 to 20, wherein the oligonucleotide comprises a hydrophobic moiety covalently attached at a 5’-terminus, 3’-terminus, or internucleoside linkage of the oligonucleotide. 22. The oligonucleotide of any one of paragraphs 1 to 21 , wherein the oligonucleotide comprises a region complementary to a coding sequence within the NRL target nucleic acid.

23. The oligonucleotide of any one of paragraphs 1 to 22, wherein the oligonucleotide comprises a total of at least 7 interlinked nucleotides.

24. The oligonucleotide of paragraph 23, wherein the single-stranded oligonucleotide comprises at least 7 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof.

25. The oligonucleotide of any one of paragraphs 1 to 24, wherein the oligonucleotide comprises a total of at least 8 interlinked nucleotides.

26. The oligonucleotide of paragraph 25, wherein the single-stranded oligonucleotide comprises at least 8 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof.

27. The oligonucleotide of any one of paragraphs 1 to 22, wherein the oligonucleotide comprises a total of at least 9 interlinked nucleotides.

28. The oligonucleotide of paragraph 22, wherein the single-stranded oligonucleotide comprises at least 9 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof.

29. The oligonucleotide of any one of paragraphs 1 to 22, wherein the oligonucleotide comprises a total of at least 10 interlinked nucleotides.

30. The oligonucleotide of paragraph 29, wherein the single-stranded oligonucleotide comprises at least 10 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof.

31 . The oligonucleotide of any one of paragraphs 1 to 30, wherein the oligonucleotide comprises a total of 10 interlinked nucleotides or fewer.

32. The oligonucleotide of any one of paragraphs 1 to 22, wherein the oligonucleotide comprises a total of at least 11 interlinked nucleotides.

33. The oligonucleotide of paragraph 32, wherein the single-stranded oligonucleotide comprises at least 11 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof.

34. The oligonucleotide of any one of paragraphs 1 to 22, wherein the oligonucleotide comprises a total of at least 12 interlinked nucleotides.

35. The oligonucleotide of paragraph 34, wherein the single-stranded oligonucleotide comprises at least 12 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof.

36. The oligonucleotide of any one of paragraphs 1 to 22, wherein the oligonucleotide comprises a total of at least 13 interlinked nucleotides.

37. The oligonucleotide of paragraph 36, wherein the single-stranded oligonucleotide comprises at least 13 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof.

38. The oligonucleotide of any one of paragraphs 1 to 22, wherein the oligonucleotide comprises a total of at least 14 interlinked nucleotides. 39. The oligonucleotide of paragraph 38, wherein the single-stranded oligonucleotide comprises at least 14 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof.

40. The oligonucleotide of any one of paragraphs 1 to 22, wherein the oligonucleotide comprises a total of at least 15 interlinked nucleotides.

41 . The oligonucleotide of paragraph 40, wherein the single-stranded oligonucleotide comprises at least 15 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof.

42. The oligonucleotide of any one of paragraphs 1 to 22, wherein the oligonucleotide comprises a total of at least 16 interlinked nucleotides.

43. The oligonucleotide of paragraph 42, wherein the single-stranded oligonucleotide comprises at least 16 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof.

44. The oligonucleotide of any one of paragraphs 1 to 43, wherein the oligonucleotide comprises a total of 25 interlinked nucleotides or fewer.

45. The oligonucleotide of any one of paragraphs 1 to 43, wherein the oligonucleotide comprises a total of 20 interlinked nucleotides or fewer.

46. The oligonucleotide of any one of paragraphs 1 to 43, wherein the oligonucleotide comprises a total of 19 interlinked nucleotides or fewer.

47. The oligonucleotide of any one of paragraphs 1 to 43, wherein the oligonucleotide comprises a total of 18 interlinked nucleotides or fewer.

48. The oligonucleotide of any one of paragraphs 1 to 43, wherein the oligonucleotide comprises a total of 17 interlinked nucleotides or fewer.

49. The oligonucleotide of any one of paragraphs 1 to 43, wherein the oligonucleotide comprises a total of 16 interlinked nucleotides or fewer.

50. The oligonucleotide of any one of paragraphs 1 to 41 , wherein the oligonucleotide comprises a total of 15 interlinked nucleotides or fewer.

51 . The oligonucleotide of any one of paragraphs 1 to 39, wherein the oligonucleotide comprises a total of 14 interlinked nucleotides or fewer.

52. The oligonucleotide of any one of paragraphs 1 to 37, wherein the oligonucleotide comprises a total of 13 interlinked nucleotides or fewer.

53. The oligonucleotide of any one of paragraphs 1 to 36, wherein the oligonucleotide comprises a total of 12 interlinked nucleotides or fewer.

54. The oligonucleotide of any one of paragraphs 1 to 33, wherein the oligonucleotide comprises a total of 11 interlinked nucleotides or fewer.

55. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 6.

56. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 7.

57. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 8. 58. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 9.

59. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 10.

60. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 11.

61 . The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 12.

62. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 13.

63. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 14.

64. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 15.

65. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 16.

66. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 17.

67. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 18.

68. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 19.

69. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 20.

70. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 21.

71 . The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 22.

72. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 23.

73. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 24.

74. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 25.

75. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 26.

76. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 27.

77. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 28.

78. The oligonucleotide of any one of paragraphs 1 to 54, wherein the nucleotide sequence is set forth in SEQ ID NO: 29. 79. The oligonucleotide of any one of paragraphs 55 to 78, wherein the sequence of the oligonucleotide comprises or consists of the nucleotide sequence referenced in the paragraph.

80. An oligonucleotide having a nucleotide sequence that comprises the sequence of any one of SEQ ID NOs: 6-29 or a chemically modified version thereof.

81. The oligonucleotide of paragraph 80, having a nucleotide sequence that consists of the sequence of any one of SEQ ID NOs: 6-29 or a chemically modified version thereof.

82. The oligonucleotide of paragraph 80 or 81 , which comprises a feature or modification as set forth in any one of paragraphs 3 to 23.

83. A pharmaceutically acceptable salt of an oligonucleotide of any one of paragraphs 1 to 82.

84. A pharmaceutical composition comprising the oligonucleotide of any one of paragraph 1 to 83 and a pharmaceutically acceptable excipient.

85. A method of inhibiting or reducing the production of an NRL protein in a cell comprising an NRL gene, the method comprising contacting the cell with the oligonucleotide of any one of paragraphs 1 to

82, the pharmaceutically acceptable salt of paragraph 83, or the pharmaceutically acceptable composition of paragraph 84.

86. The method of paragraph 85, wherein the cell is in a subject.

87. The method of paragraph 86, wherein the cell is in the subject’s eye.

88. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the oligonucleotide of any one of paragraphs 1 to 82, the pharmaceutically acceptable salt of paragraph 83, or the pharmaceutically acceptable composition of paragraph 84.

89. The method of any one of paragraphs 85 to 88, wherein the oligonucleotide or pharmaceutical composition is administered intraocularly or topically to the eye of the subject.

90. The method of any one of paragraphs 85 to 89, wherein the subject is in need of a treatment for an ocular disease, disorder, or condition associated with a dysfunction of ABCA4, AIPL1 , BBS1 , BEST1 , CEP290, CDH3, CHM, CNGA3, CNGB3, CRB1 , GUCY2D, MERTK, MRFP, MY07A, ND4, NR2E3,

PDE6, PRPH2, RD3, RHO, RLBP1 , RP1 , RPE65, RPGR, RPGRIP1 , RS1 , or SPATA7 gene.

91. The method of any one of paragraphs 85 to 90, wherein the subject is in need of a treatment for retinitis pigmentosa, Stargardt disease, cone-rod dystrophy, Leber congenital amaurosis, Bardet Biedl syndrome, macular dystrophy, dry macular degeneration, geographic atrophy, atrophic age-related macular degeneration (AMD), advanced dry AMD, retinal dystrophy, choroideremia, Usher syndrome type 1 , retinoschisis, Leber hereditary optic neuropathy, and achromatopsia.

92. The method of paragraph 91 , wherein the subject is in need of a treatment for retinitis pigmentosa.

93. The method of paragraph 92, wherein retinitis pigmentosa is Rho P23H-associated retinitis pigmentosa, PDE6-associated retinitis pigmentosa, MERTK-associated retinitis pigmentosa, BBS1- associated retinitis pigmentosa, Rho-associated retinitis pigmentosa, MRFP-associated retinitis pigmentosa, RLBP1 -associated retinitis pigmentosa, RP1 -associated retinitis pigmentosa, RPGR-X- linked retinitis pigmentosa, NR2E3-associated retinitis pigmentosa, or SPATA7-associated retinitis pigmentosa.

94. A double-stranded oligonucleotide including an oligonucleotide of paragraph 1 or 2 hybridized to a complementary oligonucleotide, wherein optionally (a) the complementary oligonucleotide has the same length as the oligonucleotide of paragraph 1 or 2, or (b) the complementary oligonucleotide has a length that is at least, e.g., ±1 , ±2, ±3, ±4, or ±5 nucleotides relative to the number of nucleotides in the oligonucleotide of paragraph 1 or 2. OTHER EMBODIMENTS

Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

Other embodiments are in the claims.

Claims

WHAT IS CLAIMED IS:

1. A single-stranded oligonucleotide comprising a total of 12 to 50 interlinked nucleotides and having a nucleobase sequence comprising at least 6 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof.

2. A single-stranded oligonucleotide comprising a total of 12 to 50 interlinked nucleotides and having a nucleobase sequence comprising at least 6 contiguous nucleobases complementary to the target sequence of any one of SEQ ID NOs: 6-17.

3. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises at least one modified nucleobase.

4. The oligonucleotide of claim 3, wherein at least one modified nucleobase is 5-methylcytosine.

5. The oligonucleotide of claim 3, wherein at least one modified nucleobase is 7-deazaguanine.

6. The oligonucleotide of claim 3, wherein at least one modified nucleobase is 6-thioguanine.

7. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises at least one modified internucleoside linkage.

8. The oligonucleotide of claim 7, wherein the modified internucleoside linkage is a phosphorothioate linkage.

9. The oligonucleotide of claim 8, wherein the phosphorothioate linkage is a stereochemically enriched phosphorothioate linkage.

10. The oligonucleotide of claim 7, wherein at least 50% of internucleoside linkages in the oligonucleotide are each independently the modified internucleoside linkage.

11 . The oligonucleotide of claim 10, wherein at least 70% of internucleoside linkages in the oligonucleotide are each independently the modified internucleoside linkage.

12. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises at least one modified sugar nucleoside.

13. The oligonucleotide of claim 12, wherein at least one modified sugar nucleoside is a bridged nucleic acid.

14. The oligonucleotide of claim 13, wherein the bridged nucleic acid is a locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), or cEt nucleic acid.

15. The oligonucleotide of claim 14, wherein the oligonucleotide is a gapmer.

16. The oligonucleotide of claim 12, wherein at least one modified sugar nucleoside is a 2’-modified sugar nucleoside.

17. The oligonucleotide of claim 16, wherein at least one 2’-modified sugar nucleoside comprises a 2’-modification selected from the group consisting of 2’-fluoro, 2’-methoxy, and 2’-methoxyethoxy.

18. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises deoxyribonucleotides.

19. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises ribonucleotides.

20. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide is a morpholino oligomer.

21 . The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a hydrophobic moiety covalently attached at a 5’-terminus, 3’-terminus, or internucleoside linkage of the oligonucleotide.

22. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a region complementary to a coding sequence within the NRL target nucleic acid.

23. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of at least 7 interlinked nucleotides.

24. The oligonucleotide of claim 23, wherein the single-stranded oligonucleotide comprises at least 7 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6- 17 and chemically modified versions thereof.

25. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of at least 8 interlinked nucleotides.

26. The oligonucleotide of claim 25, wherein the single-stranded oligonucleotide comprises at least 8 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6- 17 and chemically modified versions thereof.

27. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of at least 9 interlinked nucleotides.

28. The oligonucleotide of claim 22, wherein the single-stranded oligonucleotide comprises at least 9 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6- 17 and chemically modified versions thereof.

29. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of at least 10 interlinked nucleotides.

30. The oligonucleotide of claim 29, wherein the single-stranded oligonucleotide comprises at least

10 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof.

31 . The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of 10 interlinked nucleotides or fewer.

32. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of at least 11 interlinked nucleotides.

33. The oligonucleotide of claim 32, wherein the single-stranded oligonucleotide comprises at least

11 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof.

34. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of at least 12 interlinked nucleotides.

35. The oligonucleotide of claim 34, wherein the single-stranded oligonucleotide comprises at least

12 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof.

36. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of at least 13 interlinked nucleotides.

37. The oligonucleotide of claim 36, wherein the single-stranded oligonucleotide comprises at least

13 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof.

38. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of at least 14 interlinked nucleotides.

39. The oligonucleotide of claim 38, wherein the single-stranded oligonucleotide comprises at least

14 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof.

40. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of at least 15 interlinked nucleotides.

41 . The oligonucleotide of claim 40, wherein the single-stranded oligonucleotide comprises at least

15 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof.

42. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of at least 16 interlinked nucleotides.

43. The oligonucleotide of claim 42, wherein the single-stranded oligonucleotide comprises at least

16 contiguous nucleobases in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-17 and chemically modified versions thereof.

44. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of 25 interlinked nucleotides or fewer.

45. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of 20 interlinked nucleotides or fewer.

46. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of 19 interlinked nucleotides or fewer.

47. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of 18 interlinked nucleotides or fewer.

48. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of 17 interlinked nucleotides or fewer.

49. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of 16 interlinked nucleotides or fewer.

50. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of 15 interlinked nucleotides or fewer.

51 . The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of 14 interlinked nucleotides or fewer.

52. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of 13 interlinked nucleotides or fewer.

53. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of 12 interlinked nucleotides or fewer.

54. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises a total of 11 interlinked nucleotides or fewer.

55. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO: 6.

56. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO:

7.

57. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO:

8.

58. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO:

9.

59. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO:

10.

60. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO: 11.

61 . The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO: 12.

62. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO:

13.

63. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO:

14.

64. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO:

15.

65. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO:

16.

66. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO:

17.

67. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO:

18.

68. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO:

19.

69. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO:

20.

70. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO: 21.

71 . The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO: 22.

72. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO:

23.

73. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO:

24.

74. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO:

25.

75. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO:

26.

76. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO:

27.

77. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO:

28.

78. The oligonucleotide of claim 1 or 2, wherein the nucleotide sequence is set forth in SEQ ID NO: 29.

79. The oligonucleotide of claim 55, wherein the sequence of the oligonucleotide comprises or consists of the nucleotide sequence referenced in the claim.

80. An oligonucleotide having a nucleotide sequence that comprises the sequence of any one of SEQ ID NOs: 6-29 or a chemically modified version thereof.

81 . The oligonucleotide of claim 80, having a nucleotide sequence that consists of the sequence of any one of SEQ ID NOs: 6-29 or a chemically modified version thereof.

82. The oligonucleotide of claim 80, which comprises a feature or modification as set forth herein.

83. A pharmaceutically acceptable salt of an oligonucleotide of claim 1 or 2.

84. A pharmaceutical composition comprising the oligonucleotide of claim 1 and a pharmaceutically acceptable excipient.

85. A method of inhibiting or reducing the production of an NRL protein in a cell comprising an NRL gene, the method comprising contacting the cell with the oligonucleotide of claim 1 or 2, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising said oligonucleotide or said pharmaceutically acceptable salt.

86. The method of claim 85, wherein the cell is in a subject.

87. The method of claim 86, wherein the cell is in the subject’s eye.

88. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the oligonucleotide of claim 1 or 2, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising said oligonucleotide or said pharmaceutically acceptable salt.

89. The method of claim 85, wherein the oligonucleotide or pharmaceutical composition is administered intraocularly or topically to the eye of the subject.

90. The method of claim 85, wherein the subject is in need of a treatment for an ocular disease, disorder, or condition associated with a dysfunction of ABCA4, AIPL1 , BBS1 , BEST1 , CEP290, CDH3, CHM, CNGA3, CNGB3, CRB1 , GUCY2D, MERTK, MRFP, MY07A, ND4, NR2E3, PDE6, PRPH2, RD3, RHO, RLBP1 , RP1 , RPE65, RPGR, RPGRIP1 , RS1 , or SPATA7 gene.

91 . The method of claim 85, wherein the subject is in need of a treatment for retinitis pigmentosa, Stargardt disease, cone-rod dystrophy, Leber congenital amaurosis, Bardet Biedl syndrome, macular dystrophy, dry macular degeneration, geographic atrophy, atrophic age-related macular degeneration (AMD), advanced dry AMD, retinal dystrophy, choroideremia, Usher syndrome type 1 , retinoschisis, Leber hereditary optic neuropathy, and achromatopsia.

92. The method of claim 91 , wherein the subject is in need of a treatment for retinitis pigmentosa.

93. The method of claim 92, wherein retinitis pigmentosa is Rho P23H-associated retinitis pigmentosa, PDE6-associated retinitis pigmentosa, MERTK-associated retinitis pigmentosa, BBS1- associated retinitis pigmentosa, Rho-associated retinitis pigmentosa, MRFP-associated retinitis pigmentosa, RLBP1 -associated retinitis pigmentosa, RP1 -associated retinitis pigmentosa, RPGR-X- linked retinitis pigmentosa, NR2E3-associated retinitis pigmentosa, or SPATA7-associated retinitis pigmentosa.

94. The method of claim 88, wherein the oligonucleotide or pharmaceutical composition is administered intraocularly or topically to the eye of the subject.

95. The method claim 88, wherein the subject is in need of a treatment for an ocular disease, disorder, or condition associated with a dysfunction of ABCA4, AIPL1 , BBS1 , BEST 1 , CEP290, CDH3, CHM, CNGA3, CNGB3, CRB1 , GUCY2D, MERTK, MRFP, MY07A, ND4, NR2E3, PDE6, PRPH2, RD3, RHO, RLBP1 , RP1 , RPE65, RPGR, RPGRIP1, RS1 , or SPATA7 gene.

96. The method of claim 88, wherein the subject is in need of a treatment for retinitis pigmentosa, Stargardt disease, cone-rod dystrophy, Leber congenital amaurosis, Bardet Biedl syndrome, macular dystrophy, dry macular degeneration, geographic atrophy, atrophic age-related macular degeneration (AMD), advanced dry AMD, retinal dystrophy, choroideremia, Usher syndrome type 1 , retinoschisis,

Leber hereditary optic neuropathy, and achromatopsia.

97. The method of claim 96, wherein the subject is in need of a treatment for retinitis pigmentosa.

98. The method of claim 97, wherein retinitis pigmentosa is Rho P23H-associated retinitis pigmentosa, PDE6-associated retinitis pigmentosa, MERTK-associated retinitis pigmentosa, BBS1- associated retinitis pigmentosa, Rho-associated retinitis pigmentosa, MRFP-associated retinitis pigmentosa, RLBP1 -associated retinitis pigmentosa, RP1 -associated retinitis pigmentosa, RPGR-X- linked retinitis pigmentosa, NR2E3-associated retinitis pigmentosa, or SPATA7-associated retinitis pigmentosa.

99. A double-stranded oligonucleotide including an oligonucleotide of claim 1 or 2 hybridized to a complementary oligonucleotide, wherein optionally (a) the complementary oligonucleotide has the same length as the oligonucleotide of claim 1 or 2, or (b) the complementary oligonucleotide has a length that is at least, e.g., ±1 , ±2, ±3, ±4, or ±5 nucleotides relative to the number of nucleotides in the oligonucleotide of claim 1 or 2.