Oligonucleotides comprising PN linkages and methods thereof

Abstract

Among other things, the present disclosure provides oligonucleotides comprising PN linkages. In some embodiments, the present disclosure provides compositions comprising such oligonucleotides. In some embodiments, the present disclosure provides methods for manufacturing and using such oligonucleotides and compositions thereof. In some embodiments, the present disclosure provides methods for treating various conditions, diseases and disorders using such oligonucleotides and compositions thereof.

Description

Attorney Docket No.: 2010581-1497 OLIGONUCLEOTIDES COMPRISING PN LINKAGES AND METHODS THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application No.63 / 575,562, filed on April 5, 2024, the entirety of which is incorporated herein by reference. TECHNICAL FIELD

[0002] Among other things, the present disclosure provides oligonucleotides, compositions and methods (e.g., of preparation, use, etc.) thereof. In some embodiments, provided technologies are useful for preventing and / or treating various conditions, disorders or diseases. BACKGROUND

[0003] Oligonucleotides are useful in various applications, e.g., therapeutic, diagnostic, and / or research applications. For example, oligonucleotides targeting various genes can be useful for treatment of conditions, disorders or diseases related to such target genes. SUMMARY

[0004] Among other things, the present disclosure describes various modifications, e.g., nucleobase modifications, sugar modifications, internucleotidic linkage modifications, etc., that can be utilized to improve properties and activities of oligonucleotides, e.g., those useful for therapeutic purposes. In some embodiments, oligonucleotides comprise modified internucleotidic linkages. In some embodiments, modified internucleotidic linkages are PN linkages (e.g., those described in WO 2021237223 such as n001) which can bring various benefits and advantages for many uses. Among other things, the present disclosure demonstrates that control of stereochemistry of backbone chiral centers (stereochemistry of linkage phosphorus) and / or type of internucleotidic linkage types, optionally with control of other aspects of oligonucleotide designs, can greatly improve properties and / or activities of oligonucleotides. In some embodiments, the present disclosure provides designed oligonucleotides comprising PN internucleotidic linkages at certain positions (e.g., at certain positions in core regions of oligonucleotides comprising 5’-wing-core-wing-3’ structures) with specified linkage phosphorus stereochemistry. In some embodiments, the present disclosure demonstrates such oligonucleotides can provide improved properties and / or activities compared to reference oligonucleotides (e.g., those that do not have the same PN linkages at the same positions and / or do not have the same PN linkage phosphorus stereochemistry for such PN linkages at such positions but are otherwise identical).

[0005] For example, in some embodiments, the present disclosure provides an oligonucleotide, comprising: a PN internucleotidic linkage bonded to a sugar comprising no 2’-modification; and a PS internucleotidic linkage bonded to a sugar comprising no 2’-modification. Page 1 of 274 12621671v1Attorney Docket No.: 2010581-1497

[0006] In some embodiments, the present disclosure provides an oligonucleotide comprising: 5’−L-1−N1−L1−N2−L2−N3−L3−3’, wherein: each of N1, N2and N3is independently a nucleoside; the sugar of each of N1, N2 and N3 is independently a sugar comprising no 2’-modification; L-1is a Rp PN internucleotidic linkage; L1is a PO or Rp PS internucleotidic linkage; and each of L2and L3is independently a Sp PS internucleotidic linkage.

[0007] In some embodiments, the present disclosure provides an oligonucleotide, comprising: 5’−N-1−L-1−N1−L1−N2−L2−N3−L3−3’, wherein: each of N-1, N1, N2 and N3 is independently a nucleoside; the sugar of each of N-1, N1, N2 and N3 is independently a sugar comprising no 2’-modification; L-1is a Rp PN internucleotidic linkage; L1is a PO or Rp PS internucleotidic linkage; and each of L2and L3is independently a Sp PS internucleotidic linkage.

[0008] In some embodiments, the present disclosure provides an oligonucleotide, comprising: 5’−L-2−N-1−L-1−N1−L1−N2−L2−N3−L3−3’, wherein: each of N-1, N1, N2 and N3 is independently a nucleoside; the sugar of each of N-1, N1, N2 and N3 is independently a sugar comprising no 2’-modification; each of L-2and L-1is independently a Rp PN or Sp PS internucleotidic linkage; at least one of L-2and L-1is a Rp PN internucleotidic linkage; L1is a PO or Rp PS internucleotidic linkage; and each of L2and L3is independently a Sp PS internucleotidic linkage.

[0009] In some embodiments, the present disclosure provides an oligonucleotide, comprising: 5’−N-2−L-2−N-1−L-1−N1−L1−N2−L2−N3−L3−3’, wherein: each of N-2, N-1, N1, N2and N3is independently a nucleoside; the sugar of each of N-2, N-1, N1, N2and N3is independently a sugar comprising no 2’-modification. each of L-2and L-1is independently a Rp PN or Sp PS internucleotidic linkage; at least one of L-2and L-1is a Rp PN internucleotidic linkage; L1is a PO or Rp PS internucleotidic linkage; and each of L2and L3is independently a Sp PS internucleotidic linkage.

[0010] In some embodiments, a provided oligonucleotide comprises: 5’−L-8−N-7−L-7−N-6−L-6−N-5−L-5−N-4−L-4−N-3−L-3−N-2−L-2−N-1−L-1−N1−L1−N2−L2−N3−L3−3’, Page 2 of 274 12621671v1Attorney Docket No.: 2010581-1497 wherein: each of N-7, N-6, N-5, N-4, N-3, N-2, N-1, N1, and N2is independently a nucleoside; and each of L-8, L-7, L-6, L-5, L-4, L-3, L-2, L-1, L1, L2, and L3is independently an internucleotidic linkage.

[0011] In some embodiments, a provided oligonucleotide comprises: 5’−N-8−L-8−N-7−L-7−N-6−L-6−N-5−L-5−N-4−L-4−N-3−L-3−N-2−L-2−N-1−L-1−N1−L1−N2−L2−N3−L3−N4−3’, wherein: each of N-8, N-7, N-6, N-5, N-4, N-3, N-2, N-1, N1, N2, and N3is independently a nucleoside; and each of L-8, L-7, L-6, L-5, L-4, L-3, L-2, L-1, L1, L2, and L3is independently an internucleotidic linkage.

[0012] In some embodiments, L1is a Rp PS internucleotidic linkage. In some embodiments, L2is a Sp PS internucleotidic linkage. In some embodiments, L3is a Sp PS internucleotidic linkage. In some embodiments, L-1is a Rp PN internucleotidic linkage. In some embodiments, L-1is a Rp PN internucleotidic linkage, L1is a Rp PS internucleotidic linkage, and each of L2and L3is independently a Sp PS internucleotidic linkage.

[0013] In some embodiments, an oligonucleotide comprises 5’-wing-core-wing-3’. In some embodiments, each wing independently has a length of about 2-10 (e.g., 4-10, 5-10, 4-6, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) nucleobases. In some embodiments, each nucleobase counted in a length is independently an optionally substituted nucleobase selected from A, T, C, G, U, and tautomers of A, T, C, G and U. In some embodiments, each wing independently comprises a sugar modification. In some embodiments, each sugar in a wing is independently a modified sugar. In some embodiments, a modified sugar is a 2’-modified sugar which comprises a 2’-substituent that is different from the 2’−OH in a natural RNA sugars. In some embodiments, a 2’-modification replaces the 2’−OH in a natural DNA sugar. In some embodiments, a 2’-modification is 2’- ORsawherein R1is optionally substituted C1-6 aliphatic. In some embodiments, a 2’-modification is 2’−O−Ls2c−, wherein the oxygen atom is bonded to the 2’-carbon and Ls2cis bonded to another carbon of the sugar ring, e.g., the 4’-carbon, and Ls2cis an optionally substituted bivalent C1-2 aliphatic group. In some embodiments, Ls2cis Ls24which is optionally substituted −CH2− and is bonded to the 4’-carbon. In some embodiments, Ls24is −CH2− and is bonded to the 4’-carbon. In some embodiments, Ls24is −CH(CH3)− and is bonded to the 4’-carbon. In some embodiments, Ls24is –(R)−CH(CH3)− and is bonded to the 4’-carbon. In some embodiments, Ls24is −(S)−CH(CH3)− and is bonded to the 4’-carbon.

[0014] In some embodiments, a core has a length of about 5-50 (e.g., 5-40, 5-30, 5-20, 5-15, 5-10, 4-10, 5-10, 8-20, 9-10, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) nucleobases. In some embodiments, each nucleobase counted in a length is independently an optionally substituted nucleobase selected from A, T, C, G, U, and tautomers of A, T, C, G and U. In some embodiments, a core comprises one or more (e.g., 5-20, 8-20, 9-10, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) nucleosides, each of which independently comprises a natural DNA sugar. In some embodiments, each sugar in a core is independently a natural DNA sugar.

[0015] In some embodiments, the pattern of internucleotidic linkages of the oligonucleotide comprises (Ln)tR(S)n, wherein t is 1, 2, 3, 4, or 5, n is 2, 3, 4, 5, 6, 7 or 8, each Lnis independently R, S, nR or nS, each R Page 3 of 274 12621671v1Attorney Docket No.: 2010581-1497 independently represents a PS linkage in Rp configuration, each S independently represents a PS linkage in Sp configuration, each nR independently represents a PN linkage in Rp configuration, each nS independently represents a PN linkage in Sp configuration, and each internucleotidic linkage of the (Ln)m(Ln)tR(S)nindependently bonds to a sugar in the core. In some embodiments, at least one Lnis independently nR. In some embodiments, the pattern of internucleotidic linkages of an oligonucleotide comprises (nR)tR(S)n, wherein t is 1, 2, 3, 4, or 5, n is 2, 3, 4, 5, 6, 7 or 8, R represents a PS linkage in Rp configuration, each S independently represents a PS linkage in Sp configuration, each nR independently represents a PN linkage in Rp configuration, and each internucleotidic linkage of the (nR)tR(S)n independently bonds to a sugar in the core. In some embodiments, n is 2. In some embodiments, the pattern of internucleotidic linkages of an oligonucleotide comprises (nR)tRSS, wherein t is 0, 1, 2, 3, or 4, R represents a PS linkage in Rp configuration, each S independently represents a PS linkage in Sp configuration, each nR independently represents a PN linkage in Rp configuration, and each internucleotidic linkage of the (nR)tRSS independently bonds to a sugar in the core. In some embodiments, the pattern of internucleotidic linkages of the oligonucleotide comprises (Ln)m(Ln)tR(S)n, wherein m is 1, 2, 3, 4 or 5, t is 1, 2, 3, 4, or 5, n is 2, 3, 4, 5, 6, 7 or 8, each Lnis independently R, S, nR or nS, each R independently represents a PS linkage in Rp configuration, each S independently represents a PS linkage in Sp configuration, each nR independently represents a PN linkage in Rp configuration, each nS independently represents a PN linkage in Sp configuration, and each internucleotidic linkage of the (Ln)m(Ln)tR(S)n independently bonds to a sugar in the core. In some embodiments, the pattern of internucleotidic linkages of the oligonucleotide comprises (Ln)m(nR)tR(S)n, wherein m is 1, 2, 3, 4 or 5, t is 1, 2, 3, 4, or 5, n is 2, 3, 4, 5, 6, 7 or 8, each Lnis independently R, S, nR or nS, each R independently represents a PS linkage in Rp configuration, each S independently represents a PS linkage in Sp configuration, each nR independently represents a PN linkage in Rp configuration, each nS independently represents a PN linkage in Sp configuration, and each internucleotidic linkage of the (Ln)m(nR)tR(S)n independently bonds to a sugar in the core.

[0016] In some embodiments, an oligonucleotide comprises an internucleotidic linkage which is a PO linkage. In some embodiments, an oligonucleotide comprises an internucleotidic linkage which is a PS linkage. In some embodiments, an oligonucleotide comprises an internucleotidic linkage which is a PN linkage. In some embodiments, a PO linkage is a natural phosphate linkage. In some embodiments, each PO linkage is a natural phosphate linkage. In some embodiments, a PS linkage is a phosphorothioate linkage. In some embodiments, each PS linkage is a phosphorothioate linkage. In some embodiments, a PN linkage is a phosphoramidate linkage. In some embodiments, a PN linkage is a phosphoryl guanidine linkage. In some embodiments, a PN linkage is a n001 linkage. In some embodiments, each PN linkage is independently a phosphoramidate linkage. In some embodiments, each PN linkage is independently a phosphoryl guanidine linkage. In some embodiments, each PN linkage is a n001 linkage.

[0017] Oligonucleotides of the present disclosure can be manufactured stereoselectively. In some embodiments, each PS internucleotidic linkage is independently chirally controlled. In some embodiments, each PN internucleotidic linkage is independently chirally controlled. In some embodiments, each chiral Page 4 of 274 12621671v1Attorney Docket No.: 2010581-1497 linkage phosphorus is independently chirally controlled. In some embodiments, each PS internucleotidic linkage independently has a stereochemical purity of about or at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, each PN internucleotidic linkage independently has a stereochemical purity of about or at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, each chiral internucleotidic linkage independently has a stereochemical purity of about or at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, diastereomeric excess or enantiomeric excess of a chiral linkage phosphorus atom is about or at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, diastereomeric excess or enantiomeric excess of linkage phosphorus in each PS internucleotidic linkage is independently about or at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, diastereomeric excess or enantiomeric excess of linkage phosphorus in each PN internucleotidic linkage is independently about or at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, diastereomeric excess or enantiomeric excess of linkage phosphorus in each chiral internucleotidic linkage is independently about or at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0018] In some embodiments, an oligonucleotide composition is a chirally controlled oligonucleotide composition. In some embodiments, about or at least about 5%-100%, 10%-100%, 20-100%, 30%-100%, 40%- 100%, 50%-100%, 5%-90%, 10%-90%, 20-90%, 30%-90%, 40%-90%, 50%-90%, 5%-85%, 10%-85%, 20- 85%, 30%-85%, 40%-85%, 50%-85%, 5%-80%, 10%-80%, 20-80%, 30%-80%, 40%-80%, 50%-80%, 5%- 75%, 10%-75%, 20-75%, 30%-75%, 40%-75%, 50%-75%, 5%-70%, 10%-70%, 20-70%, 30%-70%, 40%- 70%, 50%-70%, 5%-65%, 10%-65%, 20-65%, 30%-65%, 40%-65%, 50%-65%, 5%-60%, 10%-60%, 20-60%, 30%-60%, 40%-60%, 50%-60%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in a composition that share the constitution of an oligonucleotide are the oligonucleotide. In some embodiments, the percentage of an oligonucleotide in the oligonucleotides in the composition that share the constitution of the oligonucleotide is about or at least about (DS)nc, wherein DS is about 85%-100% (e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% or more) and nc is the number of chiral internucleotidic linkages in the oligonucleotide.

[0019] In some embodiments, an oligonucleotide composition is a pharmaceutical composition. In some embodiments, an oligonucleotide is in a pharmaceutically acceptable salt form in a composition. In some embodiments, a composition comprises multiple forms of an oligonucleotide, e.g., multiple salt forms of an oligonucleotide.

[0020] Oligonucleotides described herein and compositions thereof can be utilized for various applications. In some embodiments, the present disclosure provides methods for reducing level of a RNA in a system, comprising administering or delivering to the system an effective amount of a provided oligonucleotide or a composition thereof. In some embodiments, the present disclosure provides methods for cleaving a RNA Page 5 of 274 12621671v1Attorney Docket No.: 2010581-1497 in a system, comprising administering or delivering to the system an effective amount of a provided oligonucleotide or a composition thereof. In some embodiments, certain cleavage is by RNase H. In some embodiments, the present disclosure provides methods for reducing level of a polypeptide in a system, comprising administering or delivering to the system an effective amount of a provided oligonucleotide or a composition thereof. In some embodiments, the system comprises or expresses a RNA encoding a polypeptide. In some embodiments, the base sequence of an oligonucleotide is complementary to that of the RNA or a portion thereof. In some embodiments, an oligonucleotide can hybrid to a RNA or a portion thereof when the oligonucleotide is administered or delivered to a system comprising the RNA.

[0021] Among other things, the present disclosure demonstrates that PN internucleotidic linkages can be utilized in middle portions of oligonucleotides (e.g., in core regions of oligonucleotides comprising 5’-wing- core-wing-3’ structures) to provide various benefits and advantages.

[0022] In some embodiments, the present disclosure provides a method for treating a condition, disorder or disease, comprising administering or delivering to a subject suffering therefrom an effective amount of a provided oligonucleotide or composition. In some embodiments, a condition, disorder or disease is associated with level of a RNA or a polypeptide encoded thereby. In some embodiments, the base sequence of an oligonucleotide is complementary to that of the RNA or a portion thereof. In some embodiments, an oligonucleotide can hybrid to a RNA or a portion thereof when the oligonucleotide is administered or delivered to the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1. Provided technologies can provide reduction of levels of target nucleic acid. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. iCell GABA neurons (Fujifilm Cellular Dynamics) were dosed gymnotically with either 500 nM or 50 nM of indicated oligonucleotides targeting Malat1. Cells were harvested after 4 days, and RNA was collected and levels of Malat1 mRNA and SFRS9 mRNA were quantified by qPCR. Mean % Malat1 mRNA remaining as normalized to level of SFRS9 mRNA is shown. For each oligonucleotide, the left column represents data from dosing with 500 nM of the indicated oligonucleotide and the right column represents dosing with 50 nM of the indicated oligonucleotide. N=2.

[0024] Figure 2. Provided technologies can provide reduction of levels of target nucleic acids. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. iCell GABA neurons (Fujifilm Cellular Dynamics) were dosed gymnotically with 300 nM of indicated oligonucleotides targeting Malat1. Cells were harvested after 4 days, and RNA was collected and levels of Malat1 mRNA and SFRS9 mRNA were quantified by qPCR. Mean % Malat1 mRNA remaining as normalized to level of SFRS9 mRNA is shown. N=2.

[0025] Figure 3. Provided technologies can provide reduction of levels of target nucleic acids. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof Page 6 of 274 12621671v1Attorney Docket No.: 2010581-1497 were designed and assessed. iCell GABA neurons (Fujifilm Cellular Dynamics) were dosed gymnotically with 300 nM of indicated oligonucleotides targeting Malat1. Cells were harvested after 4 days, and RNA was collected and levels of Malat1 mRNA and SFRS9 mRNA were quantified by qPCR. Mean % Malat1 mRNA remaining as normalized to level of SFRS9 mRNA is shown. N=2.

[0026] Figure 4. Provided technologies can provide reduction of levels of target nucleic acids. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. iCell GABA neurons (Fujifilm Cellular Dynamics) were dosed gymnotically with 300 nM, 100 nM, 30 nM, or 10 nM of indicated oligonucleotides targeting Malat1. Cells were harvested after 4 days, and RNA was collected and levels of Malat1 mRNA and SFRS9 mRNA were quantified by qPCR. Mean % Malat1 mRNA remaining as normalized to level of SFRS9 mRNA is shown. For each oligonucleotide, the four columns from left to right represent data from dosing with 300 nM, 100 nM, 30 nM, and 10 nM of the indicated oligonucleotide, respectively. N=2.

[0027] Figure 5. Provided technologies can provide reduction of levels of target nucleic acids. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. iCell GABA neurons (Fujifilm Cellular Dynamics) were dosed gymnotically with 300 nM, 100 nM, 30 nM, or 10 nM of indicated oligonucleotides targeting Malat1. Cells were harvested after 4 days, and RNA was collected and levels of Malat1 mRNA and SFRS9 mRNA were quantified by qPCR. Mean % Malat1 mRNA remaining as normalized to level of SFRS9 mRNA is shown. For each oligonucleotide, the four columns from left to right represent data from dosing with 300 nM, 100 nM, 30 nM, and 10 nM of the indicated oligonucleotide, respectively. N=2.

[0028] Figure 6. Provided technologies can provide reduction of levels of target nucleic acids. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. iCell GABA neurons (Fujifilm Cellular Dynamics) were dosed gymnotically with 300 nM, 100 nM, 30 nM, or 10 nM of indicated oligonucleotides targeting Malat1. Cells were harvested after 4 days, and RNA was collected and levels of Malat1 mRNA and SFRS9 mRNA were quantified by qPCR. Mean % Malat1 mRNA remaining as normalized to level of SFRS9 mRNA is shown. For each oligonucleotide, the four columns from left to right represent data from dosing with 300 nM, 100 nM, 30 nM, and 10 nM of the indicated oligonucleotide, respectively. N=2.

[0029] Figure 7. Provided technologies can provide reduction of levels of target nucleic acids. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. iCell GABA neurons (Fujifilm Cellular Dynamics) were dosed gymnotically with 150 nM, 50 nM, or 15 nM of indicated oligonucleotides targeting Malat1. Cells were harvested after 4 days, and RNA was collected and levels of Malat1 mRNA and SFRS9 mRNA were quantified by qPCR. Mean % Malat1 mRNA remaining as normalized to level of SFRS9 mRNA is shown in (A), (B), and (C). For each oligonucleotide, the three columns from left to right represent data from dosing with 150 nM, 50 nM, and 15 nM of the indicated oligonucleotide, respectively. N=2. Page 7 of 274 12621671v1Attorney Docket No.: 2010581-1497

[0030] Figure 8. Provided technologies can provide reduction of levels of target nucleic acids. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. iCell GABA neurons (Fujifilm Cellular Dynamics) were dosed gymnotically with 150 nM, 50 nM, or 15 nM of indicated oligonucleotides targeting Malat1. Cells were harvested after 4 days, and RNA was collected and levels of Malat1 mRNA and SFRS9 mRNA were quantified by qPCR. Mean % Malat1 mRNA remaining as normalized to level of SFRS9 mRNA is shown. For each oligonucleotide, the three columns from left to right represent data from dosing with 150 nM, 50 nM, and 15 nM of the indicated oligonucleotide, respectively.

[0031] Figure 9. Provided technologies can provide reduction of levels of target nucleic acids. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. iCell GABA neurons (Fujifilm Cellular Dynamics) were dosed gymnotically with 150 nM, 50 nM, or 15 nM of indicated oligonucleotides targeting Malat1. Cells were harvested after 4 days, and RNA was collected and levels of Malat1 mRNA and SFRS9 mRNA were quantified by qPCR. Mean % Malat1 mRNA remaining as normalized to level of SFRS9 mRNA is shown in (A) and (B). For each oligonucleotide, the three bars from bottom to top represent data from dosing with 150 nM, 50 nM, and 15 nM of the indicated oligonucleotide, respectively. * indicates no data shown.

[0032] Figure 10. Provided technologies can provide reduction of levels of target nucleic acids. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. iCell GABA neurons (Fujifilm Cellular Dynamics) were dosed gymnotically with 150 nM, 50 nM, or 15 nM of indicated oligonucleotides targeting Malat1. Cells were harvested after 4 days, and RNA was collected and levels of Malat1 mRNA and SFRS9 mRNA were quantified by qPCR. Mean % Malat1 mRNA remaining as normalized to level of SFRS9 mRNA is shown in (A) and (B). For each oligonucleotide, the three bars from bottom to top represent data from dosing with 150 nM, 50 nM, and 15 nM of the indicated oligonucleotide, respectively.

[0033] Figure 11. Provided technologies can reduce levels of target nucleic acids. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. Heteroduplexes were prepared by mixing equimolar solutions of indicated oligonucleotides and synthetic Malat1 RNA oligomer (5’- GAGUCAUAACCAGCCUGGCA-3’) to yield a final concentration of 20 uM. Reaction mixtures containing 5 uM heteroduplexes in RNase H buffer in a total volume of 100 uL were prepared and incubated at 37°C for 10 minutes prior to addition of human RNase H at a final concentration ratio of 400:1 (substrate : RNase H). Reactions were quenched at 5, 10, 15, 20, 30, 40, and 60 minutes by adding 10 uL of 500 mM EDTA disodium solution in water. For the 0 minutes time point, the EDTA solution was added prior to the RNase H. UV absorbance was recorded at 260, 254, 210, and 280 nm for each reaction after injection onto a column and peak areas from the resulting chromatograms corresponding to full-length RNA oligomer were integrated and normalized to the antisense strand. (A) Provided technologies can provide directed target nucleic acid cleavage. Diagrams depict heteroduplexes of indicated oligonucleotides with the Page 8 of 274 12621671v1Attorney Docket No.: 2010581-1497 synthetic Malat1 RNA oligomer and detected cleavage sites. Arrowheads and arrows indicate detected cleavage sites, wherein arrows indicate major cleavage sites. (B) Provided technologies can provide increased cleavage levels and rates. Shown is the % full-length RNA remaining at various time points. The 0 minutes time point was defined as 100% for each reaction. N=3.

[0034] Figure 12. Provided technologies can provide reduction of levels of target nucleic acids in vivo. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. Male B6 mice were orally dosed with 25 mg / kg on day 0 with the indicated oligonucleotide compositions targeting Malat1 or negative control oligonucleotide composition (ASO- 0136452), or with PBS only as a negative control. Certain oligonucleotides were GalNAc conjugated. At day 3 or day 7, animals were sacrificed and tissue samples collected. RNA was isolated from the tissue samples. Shown is % Malat1 mRNA remaining as normalized to level of HPRT mRNA as quantified by qPCR in (A) liver, (B) quadriceps, (C) stomach, (D) small intestine, (E) large intestine, (F) heart, and (G) brain. For each oligonucleotide composition and each graph, the left column represents data from day 3 and the right column represents data from day 7. N=5 per group.

[0035] Figure 13. Provided technologies can be delivered to and accumulate in target tissues in vivo. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. Male B6 mice were orally dosed with 25 mg / kg on day 0 with the indicated oligonucleotide compositions targeting Malat1 or negative control oligonucleotide composition (ASO- 0136452), or with PBS only as a negative control. Certain oligonucleotides were GalNAc conjugated. At day 3 or day 7, animals were sacrificed and tissue samples collected. RNA was isolated from the tissue samples. Accumulation of indicated oligonucleotides in (A) liver, (B) quadriceps, (C) stomach, (D) small intestine, (E) large intestine, (F) heart, and (G) brain is shown. For each oligonucleotide composition and each graph, the left column represents data from day 3 and the right column represents data from day 7. N=5 per group.

[0036] Figure 14. Provided technologies can provide reduction of levels of target nucleic acids in vivo. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. Male B6 mice were orally dosed with 25 mg / kg on day 0 with the indicated oligonucleotide compositions targeting Malat1 or negative control oligonucleotide composition (ASO- 0136452), or with PBS only as a negative control. Certain oligonucleotides were GalNAc conjugated. At day 7, animals were sacrificed and tissue samples collected. RNA was isolated from the tissue samples. Shown is % Malat1 mRNA remaining as normalized to level of HPRT mRNA as quantified by qPCR in (A) lung and (B) spleen. N=5 per group.

[0037] Figure 15. Provided technologies can provide reduction of levels of target nucleic acids in vivo. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. Male B6 mice were intranasally dosed with 25 mg / kg on day 0 with the indicated oligonucleotide compositions targeting Malat1 or negative control oligonucleotide composition (ASO- 0136452), or with PBS only as a negative control. At day 3 or day 7, animals were sacrificed and tissue samples Page 9 of 274 12621671v1Attorney Docket No.: 2010581-1497 collected. RNA was isolated from the tissue samples. Shown is % Malat1 mRNA remaining as normalized to level of HPRT mRNA as quantified by qPCR in (A) liver, (B) eye, (C) lung, (D) kidney, (E) brain, and (F) heart. N=5 per group.

[0038] Figure 16. Provided technologies can be delivered to and accumulate in target tissues in vivo. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. Male B6 mice were intranasally dosed with 25 mg / kg on day 0 with the indicated oligonucleotide compositions targeting Malat1 or negative control oligonucleotide composition (ASO- 0136452), or with PBS only as a negative control. At day 3 or day 7, animals were sacrificed and tissue samples collected. RNA was isolated from the tissue samples. Accumulation of indicated oligonucleotides in (A) liver and (B) lung is shown. N=5 per group.

[0039] Figure 17. Provided technologies can provide reduction of levels of target nucleic acids in vivo. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. Male B6 mice were subcutaneously dosed with 25 mg / kg (for oligonucleotides without GalNAc) or 1 mg / kg (for oligonucleotides with GalNAc) on day 0 with the indicated oligonucleotide compositions targeting Malat1 or negative control oligonucleotide composition (ASO-0136452), or with PBS only as a negative control. Certain oligonucleotides were GalNAc conjugated. At day 28, animals were sacrificed and tissue samples collected. RNA was isolated from the tissue samples. Shown is % Malat1 mRNA remaining as normalized to level of HPRT mRNA as quantified by qPCR in (A) liver, (B) kidney, (C) heart, (D) quadriceps, (E) small intestine, (F) large intestine, (G) brain, (H) lung, (I) stomach, and (J) spleen. N=5 per group.

[0040] Figure 18. Provided technologies can be delivered to and accumulate in target tissues in vivo. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. Male B6 mice were subcutaneously dosed with 25 mg / kg (for oligonucleotides without GalNAc) or 1 mg / kg (for oligonucleotides with GalNAc) on day 0 with the indicated oligonucleotide compositions targeting Malat1 or negative control oligonucleotide composition (ASO-0136452), or with PBS only as a negative control. Certain oligonucleotides were GalNAc conjugated. At day 28, animals were sacrificed and tissue samples collected. RNA was isolated from the tissue samples. Accumulation of indicated oligonucleotides in (A) heart and (B) quadriceps is shown. N=5 per group.

[0041] Figure 19. Provided technologies can provide reduction of levels of target nucleic acid in vivo. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. Male B6 mice were implanted with an intracerebroventricular (ICV) cannula on day 0. At day 7, the mice were dosed with 100 ug of the indicated oligonucleotide compositions targeting Malat1 or negative control oligonucleotide composition (ASO-0136452), or with PBS only as a negative control. At 4 weeks, animals were sacrificed and tissue samples collected. RNA was isolated from the tissue samples. Shown is % Malat1 expression as normalized to HPRT expression as quantified by qPCR in (A) cerebellum, (B) spinal cord, (C) anterior cortex, (D) posterior cortex, and (E) brain stem. N=5 per group. Page 10 of 274 12621671v1Attorney Docket No.: 2010581-1497

[0042] Figure 20. Provided technologies can be well-tolerated in vivo. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. Male B6 mice were implanted with an intracerebroventricular (ICV) cannula on day 0. At day 7, the mice were dosed with 100 ug of the indicated oligonucleotide compositions targeting Malat1 or negative control oligonucleotide composition (ASO-0136452), or with PBS only as a negative control. At 4 weeks, animals were sacrificed and tissue samples collected. RNA was isolated from the tissue samples. Shown is GFAP expression normalized to TUBB3 expression and further normalized relative to PBS-treated mice, as quantified by qPCR, in (A) cerebellum, (B), spinal cord, and (C) brain stem. N=5 per group.

[0043] Figure 21. Provided technologies can be well-tolerated in vivo. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. Male B6 mice were implanted with an intracerebroventricular (ICV) cannula on day 0. At day 7, the mice were dosed with 100 ug of the indicated oligonucleotide compositions targeting Malat1 or negative control oligonucleotide composition (ASO-0136452), or with PBS only as a negative control. At 4 weeks, animals were sacrificed and tissue samples collected. RNA was isolated from the tissue samples. Shown is AIF1 expression normalized to TUBB3 expression and further normalized relative to PBS-treated mice, as quantified by qPCR, in (A) cerebellum, (B), spinal cord, and (C) brain stem. N=5 per group.

[0044] Figure 22. Provided technologies can be well-tolerated in vivo. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. Male B6 mice were implanted with an intracerebroventricular (ICV) cannula on day 0. At day 7, the mice were dosed with 100 ug of the indicated oligonucleotide compositions targeting Malat1 or negative control oligonucleotide composition (ASO-0136452), or with PBS only as a negative control. At 4 weeks, animals were sacrificed and tissue samples collected. RNA was isolated from the tissue samples. Shown is (A) PCP2, (B) Grid2, or (C) Calb1 expression normalized to TUBB3 expression and further normalized relative to PBS- treated mice, as quantified by qPCR, in cerebellum. N=5 per group.

[0045] Figure 23. Provided technologies can provide reduction of levels of target nucleic acids in vivo. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. Male B6 mice were implanted with an intracerebroventricular (ICV) cannula on day 0. At day 7, the mice were dosed with 100 ug of the indicated oligonucleotide compositions targeting TARDBP, or with PBS only as a negative control. At 8 weeks, animals were sacrificed and tissue samples collected. RNA was isolated from the tissue samples. Shown is % TARDBP mRNA remaining normalized to level of TUBB3 mRNA, and further normalized relative to PBS-treated mice, as quantified by qPCR, in (A) cortex, (B), spinal cord, (C) cerebellum, and (D) brain stem. N=5 per group.

[0046] Figure 24. Provided technologies can provide reduction of levels of target nucleic acids. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. Wild-type human spinal motor neurons (BrainXell) were plated on poly-D-lysine- coated 96-well plates (Corning). At 4 days after plating, cells were dosed gymnotically with various Page 11 of 274 12621671v1Attorney Docket No.: 2010581-1497 concentrations of indicated oligonucleotides targeting TARDBP by refreshing the media with media containing the indicated oligonucleotides. Cells were harvested after 7 days, and RNA was collected and levels of TARDBP mRNA and SFRS9 mRNA were quantified by qPCR. Mean % TARDBP mRNA remaining as normalized to level of SFRS9 mRNA is shown. N=2.

[0047] Figure 25. Provided technologies can provide reduction of levels of target nucleic acids. Oligonucleotides comprising various modifications, linkage phosphorus stereochemistry and patterns thereof were designed and assessed. Wild-type human spinal motor neurons (BrainXell) were plated on poly-D-lysine- coated 96-well plates (Corning). At 4 days after plating, cells were dosed gymnotically with various concentrations of indicated oligonucleotides targeting TARDBP by refreshing the media with media containing the indicated oligonucleotides. Cells were harvested after 7 days, and RNA was collected and levels of TARDBP mRNA and SFRS9 mRNA were quantified by qPCR. Mean % TARDBP mRNA remaining as normalized to level of SFRS9 mRNA is shown. N=2. As shown in (A) to (E), provided oligonucleotides of various structural features can reduce levels of target nucleic acid. For each oligonucleotide, left: 1 uM; right: 10 uM. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0048] Technologies of the present disclosure may be understood more readily by reference to the following detailed description of certain embodiments. Definitions

[0049] As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001.

[0050] As used herein in the present disclosure, unless otherwise clear from context, (i) the term “a” or “an” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising”, “comprise”, “including” (whether used with “not limited to” or not), and “include” (whether used with “not limited to” or not) may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; (iv) the term “another” may be understood to mean at least an additional / second one or more; (v) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (vi) where ranges are provided, endpoints are included.

[0051] Unless otherwise specified, description of oligonucleotides and elements thereof (e.g., base sequence, sugar modifications, internucleotidic linkages, linkage phosphorus stereochemistry, patterns thereof, etc.) is from 5’ to 3’. As those skilled in the art will appreciate, in some embodiments, oligonucleotides may Page 12 of 274 12621671v1Attorney Docket No.: 2010581-1497 be provided and / or utilized as salt forms, particularly pharmaceutically acceptable salt forms, e.g., sodium salts. As those skilled in the art will also appreciate, in some embodiments, individual oligonucleotides within a composition may be considered to be of the same constitution and / or structure even though, within such composition (e.g., a liquid composition), particular such oligonucleotides might be in different salt form(s) (and may be dissolved and the oligonucleotide chain may exist as an anion form when, e.g., in a liquid composition) at a particular moment in time. For example, those skilled in the art will appreciate that, at a given pH, individual internucleotidic linkages along an oligonucleotide chain may be in an acid (H) form, or in one of a plurality of possible salt forms (e.g., a sodium salt, or a salt of a different cation, depending on which ions might be present in the preparation or composition), and will understand that, so long as their acid forms (e.g., replacing all cations, if any, with H+) are of the same constitution and / or structure, such individual oligonucleotides may properly be considered to be of the same constitution and / or structure.

[0052] Aliphatic: As used herein, “aliphatic” means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation (but not aromatic), or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is completely saturated or that contains one or more units of unsaturation (but not aromatic), or combinations thereof. In some embodiments, aliphatic groups contain 1-50 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-20 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-9 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-7 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms. Certain aliphatic groups include linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0053] Alkenyl: As used herein, the term “alkenyl” refers to an aliphatic group, as defined herein, having one or more double bonds.

[0054] Alkyl: As used herein, the term “alkyl” is given its ordinary meaning in the art and refers to an aliphatic group, e.g., a straight-chain alkyl group, a branched-chain alkyl group, a cycloalkyl (alicyclic) group, an alkyl substituted cycloalkyl group, a cycloalkyl substituted alkyl group, etc., which is saturated. In some embodiments, alkyl has 1-100 carbon atoms. In some embodiments, alkyl has about 1-30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 1-25, 1-20, 1-15, 1-10, 3-30, etc.) carbon atoms. In some embodiments, alkyl has 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1-15, 1-10, etc.) carbon atoms. In certain embodiments, a straight chain or branched chain alkyl has about 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1-15, 1-10, etc.) carbon atoms in its backbone (e.g., C1-C20for straight chain, C2-C20for branched chain, etc.), and alternatively, about 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1-5, 1-4, etc.) carbon atoms. In some embodiments, Page 13 of 274 12621671v1Attorney Docket No.: 2010581-1497 cycloalkyl rings have from about 3-10 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 3-8, 3-7, 3-6, 4-6, 5-6, 5-8, etc.) carbon atoms in their ring structure where such rings are monocyclic, bicyclic, or polycyclic, and alternatively about 5, 6 or 7 carbons in the ring structure. In some embodiments, an alkyl group may be a lower alkyl group, wherein a lower alkyl group has 1-4 carbon atoms (e.g., C1-C4for straight chain lower alkyls).

[0055] Alkynyl: As used herein, the term “alkynyl” refers to an aliphatic group, as defined herein, having one or more triple bonds.

[0056] Analog: The term “analog” includes any chemical moiety which differs structurally from a reference chemical moiety or class of moieties, but which is capable of performing at least one function of such a reference chemical moiety or class of moieties. As non-limiting examples, a nucleotide analog differs structurally from a nucleotide but performs at least one function of a nucleotide; a nucleobase analog differs structurally from a nucleobase but performs at least one function of a nucleobase; etc.

[0057] Aryl: The term “aryl”, as used herein, used alone or as part of a larger moiety as in “aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic, bicyclic or polycyclic ring systems having a total of, unless otherwise specified, 5-30 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 5-25, 5-20, 5-15, 5-14, 5-10, 5-9, etc.) ring members, wherein at least one ring in the system is aromatic. In some embodiments, an aryl group is a monocyclic, bicyclic or polycyclic ring system having a total of five to fourteen (e.g., 5-10, 6-14, 6-10, 5, 6, 9, 10, 14, etc.) ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 7 ring members. In some embodiments, each monocyclic ring unit is aromatic. In some embodiments, each aromatic ring is independently 6-membered. In some embodiments, each aromatic ring atom is carbon. In some embodiments, each ring atom is carbon. In some embodiments, an aryl group is a biaryl group. The term “aryl” may be used interchangeably with the term “aryl ring.” In certain embodiments of the present disclosure, “aryl” refers to an aromatic ring system which includes, but is not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term “aryl,” as it is used herein, is a group in which an aromatic ring is fused to one or more non–aromatic rings, such as indanyl, phthalimidyl, naphthylmethyl, phenanthridinyl, or tetrahydronaphthyl, and the like.

[0058] Characteristic portion: As used herein, the term “characteristic portion”, in the broadest sense, refers to a portion of a substance whose presence (or absence) correlates with presence (or absence) of a particular feature, attribute, or activity of the substance. In some embodiments, a characteristic portion of a substance is a portion that is found in the substance and in related substances that share the particular feature, attribute or activity, but not in those that do not share the particular feature, attribute or activity. In certain embodiments, a characteristic portion shares at least one functional characteristic with the intact substance. For example, in some embodiments, a “characteristic portion” of a protein or polypeptide is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of a protein or polypeptide. In some embodiments, each such continuous stretch generally contains at least 2, 5, 10, 15, 20, 50, or more amino acids. In general, a characteristic portion of a substance Page 14 of 274 12621671v1Attorney Docket No.: 2010581-1497 (e.g., of a protein, antibody, etc.) is one that, in addition to the sequence and / or structural identity specified above, shares at least one functional characteristic with the relevant intact substance. In some embodiments, a characteristic portion may be biologically active.

[0059] Chiral control: As used herein, “chiral control” refers to control of the stereochemical designation of the chiral linkage phosphorus in a chiral internucleotidic linkage within an oligonucleotide. As used herein, a chiral internucleotidic linkage is an internucleotidic linkage whose linkage phosphorus is chiral. In some embodiments, a control is achieved through a chiral element that is absent from the sugar and base moieties of an oligonucleotide, for example, in some embodiments, a control is achieved through use of one or more chiral auxiliaries during oligonucleotide preparation, which chiral auxiliaries often are part of chiral phosphoramidites used during oligonucleotide preparation. In contrast to chiral control, a person having ordinary skill in the art will appreciate that conventional oligonucleotide synthesis which does not use chiral auxiliaries cannot control stereochemistry at a chiral internucleotidic linkage if such conventional oligonucleotide synthesis is used to form the chiral internucleotidic linkage. In some embodiments, the stereochemical designation of each chiral linkage phosphorus in each chiral internucleotidic linkage within an oligonucleotide is controlled.

[0060] Chirally controlled oligonucleotide composition: The terms “chirally controlled oligonucleotide composition”, “chirally controlled nucleic acid composition”, and the like, as used herein, refers to a composition that comprises a plurality of oligonucleotides (or nucleic acids) which share a common base sequence, wherein the plurality of oligonucleotides (or nucleic acids) share the same linkage phosphorus stereochemistry at one or more chiral internucleotidic linkages (chirally controlled or stereodefined internucleotidic linkages, whose chiral linkage phosphorus is Rp or Sp in the composition (“stereodefined”), not a random Rp and Sp mixture as non-chirally controlled internucleotidic linkages). In some embodiments, a chirally controlled oligonucleotide composition comprises a plurality of oligonucleotides (or nucleic acids) that share: 1) a common base sequence, 2) a common pattern of backbone linkages, and 3) a common pattern of backbone phosphorus modifications, wherein the plurality of oligonucleotides (or nucleic acids) share the same linkage phosphorus stereochemistry at one or more chiral internucleotidic linkages (chirally controlled or stereodefined internucleotidic linkages, whose chiral linkage phosphorus is Rp or Sp in the composition (“stereodefined”), not a random Rp and Sp mixture as non-chirally controlled internucleotidic linkages). Level of the plurality of oligonucleotides (or nucleic acids) in a chirally controlled oligonucleotide composition is pre-determined / controlled or enriched (e.g., through chirally controlled oligonucleotide preparation to stereoselectively form one or more chiral internucleotidic linkages) compared to a random level in a non- chirally controlled oligonucleotide composition. In some embodiments, about 1%-100%, (e.g., about 5%- 100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90- 100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition are oligonucleotides of the plurality. In some embodiments, about 1%-100%, Page 15 of 274 12621671v1Attorney Docket No.: 2010581-1497 (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition that share the common base sequence, the common pattern of backbone linkages, and the common pattern of backbone phosphorus modifications are oligonucleotides of the plurality. In some embodiments, a level is about 1%-100%, (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a composition, or of all oligonucleotides in a composition that share a common base sequence (e.g., of a plurality of oligonucleotide or an oligonucleotide type), or of all oligonucleotides in a composition that share a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone phosphorus modifications, or of all oligonucleotides in a composition that share a common base sequence, a common pattern of base modifications, a common pattern of sugar modifications, a common pattern of internucleotidic linkage types, and / or a common pattern of internucleotidic linkage modifications. In some embodiments, the plurality of oligonucleotides share the same stereochemistry at about 1-50 (e.g., about 1-10, 1-20, 5-10, 5-20, 10-15, 10-20, 10-25, 10-30, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) chiral internucleotidic linkages. In some embodiments, the plurality of oligonucleotides share the same stereochemistry at about 1%-100% (e.g., about 5%-100%, 10%-100%, 20%- 100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) of chiral internucleotidic linkages. In some embodiments, oligonucleotides (or nucleic acids) of a plurality share the same pattern of sugar and / or nucleobase modifications, in any. In some embodiments, oligonucleotides (or nucleic acids) of a plurality are various forms of the same oligonucleotide (e.g., acid and / or various salts of the same oligonucleotide). In some embodiments, oligonucleotides (or nucleic acids) of a plurality are of the same constitution. In some embodiments, level of the oligonucleotides (or nucleic acids) of the plurality is about 1%-100%, (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%- 100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides (or nucleic acids) in a composition that share the same constitution as the oligonucleotides (or nucleic acids) of the plurality. In some embodiments, each chiral internucleotidic linkage is a chiral controlled internucleotidic linkage, and the composition is a completely chirally controlled Page 16 of 274 12621671v1Attorney Docket No.: 2010581-1497 oligonucleotide composition. In some embodiments, oligonucleotides (or nucleic acids) of a plurality are structurally identical. In some embodiments, a chirally controlled internucleotidic linkage has a diastereopurity of about or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, typically about or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%. In some embodiments, a chirally controlled internucleotidic linkage has a diastereopurity of about or at least about 95%. In some embodiments, a chirally controlled internucleotidic linkage has a diastereopurity of about or at least about 96%. In some embodiments, a chirally controlled internucleotidic linkage has a diastereopurity of about or at least about 97%. In some embodiments, a chirally controlled internucleotidic linkage has a diastereopurity of about or at least about 98%. In some embodiments, a chirally controlled internucleotidic linkage has a diastereopurity of about or at least about 99%. In some embodiments, a percentage of a level is or is about or at least about (DS)nc, wherein DS is a diastereopurity as described in the present disclosure (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% or more) and nc is the number of chiral linkage phosphorus as described in the present disclosure (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 5-50, 5-40, 5-30, 5-25, 5- 20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more). In some embodiments, a percentage of a level is or is at least (DS)nc, wherein DS is a diastereopurity as described in the present disclosure (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% or more) and nc is the number of chirally controlled internucleotidic linkages as described in the present disclosure (e.g., 1- 50, 1-40, 1-30, 1-25, 1-20, 5-50, 5-40, 5-30, 5-25, 5-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more). In some embodiments, a percentage of a level is or is at least (DS)nc, wherein DS is about 95%-100%. For example, when DS is 99% and nc is 10, the percentage is or is at least 90% ((99%)10≈ 0.90 = 90%). In some embodiments, level of a plurality of oligonucleotides in a composition is represented as the product of the diastereopurity of each chiral linkage phosphorus. In some embodiments, level of a plurality of oligonucleotides in a composition is represented as the product of the diastereopurity of each chirally controlled internucleotidic linkage in the oligonucleotides. In some embodiments, diastereopurity of an internucleotidic linkage connecting two nucleosides in an oligonucleotide (or nucleic acid) is represented by the diastereopurity of an internucleotidic linkage of a dimer connecting the same two nucleosides, wherein the dimer is prepared using comparable conditions, in some instances, identical synthetic cycle conditions (e.g., for the linkage between Nx and Ny in an oligonucleotide ….NxNy….., the dimer is NxNy). In some embodiments, not all chiral internucleotidic linkages are chiral controlled internucleotidic linkages, and the composition is a partially chirally controlled oligonucleotide composition. In some embodiments, a non- chirally controlled internucleotidic linkage has a diastereopurity of less than about 80%, 75%, 70%, 65%, 60%, 55%, or of about 50%, as typically observed in stereorandom oligonucleotide compositions (e.g., as appreciated by those skilled in the art, from traditional oligonucleotide synthesis, e.g., the phosphoramidite method). In some embodiments, oligonucleotides (or nucleic acids) of a plurality are of the same type. In some embodiments, a chirally controlled oligonucleotide composition comprises non-random or controlled levels of individual oligonucleotide or nucleic acids types. For instance, in some embodiments a chirally controlled Page 17 of 274 12621671v1Attorney Docket No.: 2010581-1497 oligonucleotide composition comprises one and no more than one oligonucleotide type. In some embodiments, a chirally controlled oligonucleotide composition comprises more than one oligonucleotide type. In some embodiments, a chirally controlled oligonucleotide composition comprises multiple oligonucleotide types. In some embodiments, a chirally controlled oligonucleotide composition is a composition of oligonucleotides of an oligonucleotide type, which composition comprises a non-random or controlled level of a plurality of oligonucleotides of the oligonucleotide type.

[0061] Comparable: The term “comparable” is used herein to describe two (or more) sets of conditions or circumstances that are sufficiently similar to one another to permit comparison of results obtained or phenomena observed. In some embodiments, comparable sets of conditions or circumstances are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will appreciate that sets of conditions are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under the different sets of conditions or circumstances are caused by or indicative of the variation in those features that are varied.

[0062] Cycloaliphatic: The term “cycloaliphatic,” “carbocycle,” “carbocyclyl,” “carbocyclic radical,” and “carbocyclic ring,” are used interchangeably, and as used herein, refer to saturated or partially unsaturated, but non-aromatic, cyclic aliphatic monocyclic, bicyclic, or polycyclic ring systems, as described herein, having, unless otherwise specified, from 3 to 30 ring members. Cycloaliphatic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, a cycloaliphatic group has 3–6 carbons. In some embodiments, a cycloaliphatic group is saturated and is cycloalkyl. The term “cycloaliphatic” may also include aliphatic rings that are fused to one or more aromatic or nonaromatic rings, such as decahydronaphthyl or tetrahydronaphthyl. In some embodiments, a cycloaliphatic group is bicyclic. In some embodiments, a cycloaliphatic group is tricyclic. In some embodiments, a cycloaliphatic group is polycyclic. In some embodiments, “cycloaliphatic” refers to C3-C6 monocyclic hydrocarbon, or C8-C10 bicyclic or polycyclic hydrocarbon, that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule, or a C9-C16 polycyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule.

[0063] Heteroaliphatic: The term “heteroaliphatic”, as used herein, is given its ordinary meaning in the art and refers to aliphatic groups as described herein in which one or more carbon atoms are independently replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, and the like). In some embodiments, one or more units selected from C, CH, CH2, and CH3are independently replaced by one or more heteroatoms (including oxidized and / or substituted forms thereof). In some embodiments, a heteroaliphatic group is heteroalkyl. In some embodiments, a heteroaliphatic group is heteroalkenyl.

[0064] Heteroalkyl: The term “heteroalkyl”, as used herein, is given its ordinary meaning in the art and Page 18 of 274 12621671v1Attorney Docket No.: 2010581-1497 refers to alkyl groups as described herein in which one or more carbon atoms are independently replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, and the like). Examples of heteroalkyl groups include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, etc.

[0065] Heteroaryl: The terms “heteroaryl” and “heteroar–”, as used herein, used alone or as part of a larger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer to monocyclic, bicyclic or polycyclic ring systems having a total of, unless otherwise specified, 5-30 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 5-25, 5-20, 5-15, 5-14, 5-10, 5-9, etc.) ring members, wherein at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. In some embodiments, the number of ring heteroatoms in a heteroaryl group is about 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1-5, 1-4, 1-3, 1-2, etc.). In some embodiments, a heteroaryl group is a group having 5 to 10 ring atoms, in some embodiments 5, 6, 9, or 10 ring atoms. In some embodiments, each monocyclic ring unit is aromatic. In some embodiments, each aromatic monocyclic ring unit is independently 5- or 6-membered. In some embodiments, a heteroaryl ring is monocyclic and is 5-membered. In some embodiments, a heteroaryl ring is monocyclic and is 6- membered. In some embodiments, a heteroaryl ring is bicyclic and is 9-membered. In some embodiments, a heteroaryl ring is bicyclic and is 10-membered. In some embodiments, a heteroaryl group has 6, 10, or 14 π electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. In some embodiments, each heteroatom is independently selected from nitrogen, oxygen and sulfur. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. In some embodiments, a heteroaryl is a heterobiaryl group, such as bipyridyl and the like. The terms “heteroaryl” and “heteroar–”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H–quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3–b]– 1,4–oxazin–3(4H)–one. A heteroaryl group may be monocyclic, bicyclic or polycyclic. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring,” “heteroaryl group,” or “heteroaromatic,” any of which terms include rings that are optionally substituted. The term “heteroaralkyl” refers to an alkyl group substituted by a heteroaryl group, wherein the alkyl and heteroaryl portions independently are optionally substituted.

[0066] Heteroatom: The term “heteroatom", as used herein, means an atom that is not carbon or hydrogen. In some embodiments, a heteroatom is boron, oxygen, sulfur, nitrogen, phosphorus, or silicon (including oxidized forms of nitrogen, sulfur, phosphorus, or silicon; charged forms of nitrogen (e.g., quaternized forms, forms as in iminium groups, etc.), phosphorus, sulfur, oxygen; etc.). In some embodiments, a heteroatom is Page 19 of 274 12621671v1Attorney Docket No.: 2010581-1497 silicon, phosphorus, oxygen, sulfur or nitrogen. In some embodiments, a heteroatom is silicon, oxygen, sulfur or nitrogen. In some embodiments, a heteroatom is oxygen, sulfur or nitrogen.

[0067] Heterocycle: As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclic radical,” and “heterocyclic ring", as used herein, are used interchangeably and refer to a monocyclic, bicyclic or polycyclic ring moiety (unless otherwise specified, 3-30 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 3-25, 3-20, 3-15, 3-14, 3-10, 5-10, 3-7, 3-6, etc.) membered) that is saturated or partially unsaturated and has one or more (e.g., 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) heteroatom ring atoms. In some embodiments, the number of ring heteroatoms in a heterocyclyl group is about 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1-5, 1-4, 1-3, 1-2, etc.). In some embodiments, a heterocyclyl group is a stable 3-7 membered monocyclic or 7-10 membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above. In some embodiments, each monocyclic ring is independently 3-10 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 3-8, 3-7, 3-6, 4-6, 5-6, 5-8, etc.) membered. In some embodiments, each monocyclic ring independently has 0-5 (e.g., 0, 1, 2, 3, 4, 5, 1-5, 1-4, 1-2, etc.) heteroatoms wherein at least one monocyclic ring independently has 1-5 (e.g., 1, 2, 3, 4, 5, 1-5, 1-4, 1-2, etc.) heteroatoms. In some embodiments, each monocyclic ring is independently 3-7 membered. In some embodiments, each heteroatom is independently selected from nitrogen, oxygen and sulfur. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0–3 heteroatoms selected from oxygen, sulfur and nitrogen, the nitrogen may be N (as in 3,4–dihydro–2H–pyrrolyl), NH (as in pyrrolidinyl), or+NR (as in N–substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclic moiety,” and “heterocyclic radical,” are used interchangeably herein, and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H–indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. A heterocyclyl group may be monocyclic, bicyclic or polycyclic. The term “heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

[0068] Linkage phosphorus: as defined herein, the phrase “linkage phosphorus” is used to indicate that the particular phosphorus atom being referred to is the phosphorus atom present in the internucleotidic linkage, which phosphorus atom corresponds to the phosphorus atom of a phosphodiester internucleotidic linkage as occurs in naturally occurring DNA and RNA. In some embodiments, a linkage phosphorus atom is in a modified internucleotidic linkage, wherein each oxygen atom of a phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, a linkage phosphorus atom Page 20 of 274 12621671v1Attorney Docket No.: 2010581-1497 is chiral (e.g., as in phosphorothioate internucleotidic linkages). In some embodiments, a linkage phosphorus atom is achiral (e.g., as in natural phosphate linkages).

[0069] Modified nucleobase: The terms "modified nucleobase", "modified base" and the like refer to a chemical moiety which is chemically distinct from a nucleobase, but which is capable of performing at least one function of a nucleobase. In some embodiments, a modified nucleobase is a nucleobase which comprises a modification. In some embodiments, a modified nucleobase is capable of at least one function of a nucleobase, e.g., forming a moiety in a polymer capable of base-pairing to a nucleic acid comprising an at least complementary sequence of bases. In some embodiments, a modified nucleobase is substituted A, T, C, G, or U, or a substituted tautomer of A, T, C, G, or U. In some embodiments, a modified nucleobase in the context of oligonucleotides refer to a nucleobase that is not A, T, C, G or U.

[0070] Modified nucleoside: The term "modified nucleoside" refers to a moiety derived from or chemically similar to a natural nucleoside, but which comprises a chemical modification which differentiates it from a natural nucleoside. Non-limiting examples of modified nucleosides include those which comprise a modification at the base and / or the sugar. Non-limiting examples of modified nucleosides include those with a 2’ modification at a sugar. Non-limiting examples of modified nucleosides also include abasic nucleosides (which lack a nucleobase). In some embodiments, a modified nucleoside is capable of at least one function of a nucleoside, e.g., forming a moiety in a polymer capable of base-pairing to a nucleic acid comprising an at least complementary sequence of bases.

[0071] Modified nucleotide: The term “modified nucleotide” includes any chemical moiety which differs structurally from a natural nucleotide but is capable of performing at least one function of a natural nucleotide. In some embodiments, a modified nucleotide comprises a modification at a sugar, base and / or internucleotidic linkage. In some embodiments, a modified nucleotide comprises a modified sugar, modified nucleobase and / or modified internucleotidic linkage. In some embodiments, a modified nucleotide is capable of at least one function of a nucleotide, e.g., forming a subunit in a polymer capable of base-pairing to a nucleic acid comprising an at least complementary sequence of bases.

[0072] Modified sugar: The term “modified sugar” refers to a moiety that can replace a sugar. A modified sugar mimics the spatial arrangement, electronic properties, or some other physicochemical property of a sugar. In some embodiments, as described in the present disclosure, a modified sugar is substituted ribose or deoxyribose. In some embodiments, a modified sugar comprises a 2’-modification. Examples of useful 2’- modification are widely utilized in the art and described herein. In some embodiments, a 2’-modification is 2’- F. In some embodiments, a 2’-modification is 2’-OR, wherein R is optionally substituted C1-10aliphatic. In some embodiments, a 2’-modification is 2’-OMe. In some embodiments, a 2’-modification is 2’-MOE. In some embodiments, a modified sugar is a bicyclic sugar (e.g., a sugar used in LNA, BNA, etc.). In some embodiments, in the context of oligonucleotides, a modified sugar is a sugar that is not ribose or deoxyribose as typically found in natural RNA or DNA.

[0073] Nucleic acid: The term “nucleic acid”, as used herein, includes any nucleotides and polymers Page 21 of 274 12621671v1Attorney Docket No.: 2010581-1497 thereof. The term “polynucleotide”, as used herein, refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) or a combination thereof. These terms refer to the primary structure of the molecules and, thus, include double- and single-stranded DNA, and double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA comprising modified nucleotides and / or modified polynucleotides, such as, though not limited to, methylated, protected and / or capped nucleotides or polynucleotides. The terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and / or modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified internucleotidic linkages. The term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified internucleotidic linkages. Examples include, and are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. Unless otherwise specified, the prefix poly- refers to a nucleic acid containing 2 to about 10,000 nucleotide monomer units and wherein the prefix oligo- refers to a nucleic acid containing 2 to about 200 nucleotide monomer units.

[0074] Nucleobase: The term “nucleobase” refers to the parts of nucleic acids that are involved in the hydrogen-bonding that binds one nucleic acid strand to another complementary strand in a sequence specific manner. The most common naturally-occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, a naturally-occurring nucleobases are modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, a naturally-occurring nucleobases are methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, a nucleobase comprises a heteroaryl ring wherein a ring atom is nitrogen, and when in a nucleoside, the nitrogen is bonded to a sugar moiety. In some embodiments, a nucleobase comprises a heterocyclic ring wherein a ring atom is nitrogen, and when in a nucleoside, the nitrogen is bonded to a sugar moiety. In some embodiments, a nucleobase is a “modified nucleobase,” a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, a modified nucleobase is substituted A, T, C, G or U. In some embodiments, a modified nucleobase is a substituted tautomer of A, T, C, G, or U. In some embodiments, a modified nucleobases is methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, a modified nucleobase mimics the spatial arrangement, electronic properties, or some other physicochemical property of the nucleobase and retains the property of hydrogen-bonding that binds one nucleic acid strand to another in a sequence specific manner. In some embodiments, a modified nucleobase can pair with all of the five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of the oligonucleotide duplex. As used herein, the term “nucleobase” also encompasses structural analogs used in lieu of natural or naturally-occurring nucleotides, such as modified nucleobases and nucleobase analogs. In some embodiments, a nucleobase is optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U. In some Page 22 of 274 12621671v1Attorney Docket No.: 2010581-1497 embodiments, a “nucleobase” refers to a nucleobase unit in an oligonucleotide or a nucleic acid (e.g., A, T, C, G or U as in an oligonucleotide or a nucleic acid).

[0075] Nucleoside: The term “nucleoside” refers to a moiety wherein a nucleobase or a modified nucleobase is covalently bound to a sugar or a modified sugar. In some embodiments, a nucleoside is a natural nucleoside, e.g., adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, or deoxycytidine. In some embodiments, a nucleoside is a modified nucleoside, e.g., a substituted natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. In some embodiments, a nucleoside is a modified nucleoside, e.g., a substituted tautomer of a natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. In some embodiments, a “nucleoside” refers to a nucleoside unit in an oligonucleotide or a nucleic acid.

[0076] Nucleotide: The term “nucleotide” as used herein refers to a monomeric unit of a polynucleotide that consists of a nucleobase, a sugar, and one or more internucleotidic linkages (e.g., phosphate linkages in natural DNA and RNA). The naturally occurring bases [guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and uracil (U)] are derivatives of purine or pyrimidine, though it should be understood that naturally and non- naturally occurring base analogs are also included. The naturally occurring sugar is the pentose (five-carbon sugar) deoxyribose (which forms DNA) or ribose (which forms RNA), though it should be understood that naturally and non-naturally occurring sugar analogs are also included. Nucleotides are linked via internucleotidic linkages to form nucleic acids, or polynucleotides. Many internucleotidic linkages are known in the art (such as, though not limited to, phosphate, phosphorothioates, boranophosphates and the like). Artificial nucleic acids include PNAs (peptide nucleic acids), phosphotriesters, phosphorothionates, H- phosphonates, phosphoramidates, boranophosphates, methylphosphonates, phosphonoacetates, thiophosphonoacetates and other variants of the phosphate backbone of native nucleic acids, such as those described herein. In some embodiments, a natural nucleotide comprises a naturally occurring base, sugar and internucleotidic linkage. As used herein, the term “nucleotide” also encompasses structural analogs used in lieu of natural or naturally-occurring nucleotides, such as modified nucleotides and nucleotide analogs. In some embodiments, a “nucleotide” refers to a nucleotide unit in an oligonucleotide or a nucleic acid.

[0077] Oligonucleotide: The term "oligonucleotide" refers to a polymer or oligomer of nucleotides, and may contain any combination of natural and non-natural nucleobases, sugars, and internucleotidic linkages.

[0078] Oligonucleotides can be single-stranded or double-stranded. A single-stranded oligonucleotide can have double-stranded regions (formed by two portions of the single-stranded oligonucleotide) and a double- stranded oligonucleotide, which comprises two oligonucleotide chains, can have single-stranded regions for example, at regions where the two oligonucleotide chains are not complementary to each other. Example oligonucleotides include, but are not limited to structural genes, genes including control and termination regions, self-replicating systems such as viral or plasmid DNA, single-stranded and double-stranded RNAi agents and other RNA interference reagents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, Page 23 of 274 12621671v1Attorney Docket No.: 2010581-1497 ribozymes, microRNAs, microRNA mimics, supermirs, aptamers, antimirs, antagomirs, Ul adaptors, triplex- forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immuno-stimulatory oligonucleotides, and decoy oligonucleotides.

[0079] Oligonucleotides of the present disclosure can be of various lengths. In some embodiments, oligonucleotides can range from about 2 to about 200 nucleosides in length. In various related embodiments, oligonucleotides, single-stranded, double-stranded, or triple-stranded, can range in length from about 4 to about 10 nucleosides, from about 10 to about 50 nucleosides, from about 20 to about 50 nucleosides, from about 15 to about 30 nucleosides, from about 20 to about 30 nucleosides in length. In some embodiments, an oligonucleotide is from about 9 to about 39 nucleosides in length. In some embodiments, an oligonucleotide is from about 25 to about 70 nucleosides in length. In some embodiments, an oligonucleotide is from about 26 to about 70 nucleosides in length. In some embodiments, an oligonucleotide is from about 27 to about 70 nucleosides in length. In some embodiments, an oligonucleotide is from about 28 to about 70 nucleosides in length. In some embodiments, an oligonucleotide is from about 29 to about 70 nucleosides in length. In some embodiments, an oligonucleotide is from about 30 to about 70 nucleosides in length. In some embodiments, an oligonucleotide is from about 31 to about 70 nucleosides in length. In some embodiments, an oligonucleotide is from about 32 to about 70 nucleosides in length. In some embodiments, an oligonucleotide is from about 25 to about 60 nucleosides in length. In some embodiments, an oligonucleotide is from about 25 to about 50 nucleosides in length. In some embodiments, an oligonucleotide is from about 25 to about 40 nucleosides in length. In some embodiments, an oligonucleotide is from about 30 to about 40 nucleosides in length. In some embodiments, the oligonucleotide is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides in length. In some embodiments, an oligonucleotide is at least 4 nucleosides in length. In some embodiments, an oligonucleotide is at least 5 nucleosides in length. In some embodiments, an oligonucleotide is at least 6 nucleosides in length. In some embodiments, an oligonucleotide is at least 7 nucleosides in length. In some embodiments, an oligonucleotide is at least 8 nucleosides in length. In some embodiments, an oligonucleotide is at least 9 nucleosides in length. In some embodiments, an oligonucleotide is at least 10 nucleosides in length. In some embodiments, an oligonucleotide is at least 11 nucleosides in length. In some embodiments, an oligonucleotide is at least 12 nucleosides in length. In some embodiments, an oligonucleotide is at least 15 nucleosides in length. In some embodiments, an oligonucleotide is at least 15 nucleosides in length. In some embodiments, an oligonucleotide is at least 16 nucleosides in length. In some embodiments, an oligonucleotide is at least 17 nucleosides in length. In some embodiments, an oligonucleotide is at least 18 nucleosides in length. In some embodiments, an oligonucleotide is at least 19 nucleosides in length. In some embodiments, an oligonucleotide is at least 20 nucleosides in length. In some embodiments, an oligonucleotide is at least 25 nucleosides in length. In some embodiments, an oligonucleotide is at least 26 nucleosides in length. In some embodiments, an oligonucleotide is at least 27 nucleosides in length. In some embodiments, an oligonucleotide is at least 28 nucleosides in length. In some embodiments, an oligonucleotide is at least 29 nucleosides in length. In some embodiments, an oligonucleotide is at least 30 nucleosides in Page 24 of 274 12621671v1Attorney Docket No.: 2010581-1497 length. In some embodiments, an oligonucleotide is at least 31 nucleosides in length. In some embodiments, an oligonucleotide is at least 32 nucleosides in length. In some embodiments, an oligonucleotide is at least 33 nucleosides in length. In some embodiments, an oligonucleotide is at least 34 nucleosides in length. In some embodiments, an oligonucleotide is at least 35 nucleosides in length. In some embodiments, an oligonucleotide is at least 36 nucleosides in length. In some embodiments, an oligonucleotide is at least 37 nucleosides in length. In some embodiments, an oligonucleotide is at least 38 nucleosides in length. In some embodiments, an oligonucleotide is at least 39 nucleosides in length. In some embodiments, an oligonucleotide is at least 40 nucleosides in length. In some embodiments, an oligonucleotide is 25 nucleosides in length. In some embodiments, an oligonucleotide is 26 nucleosides in length. In some embodiments, an oligonucleotide is 27 nucleosides in length. In some embodiments, an oligonucleotide is 28 nucleosides in length. In some embodiments, an oligonucleotide is 29 nucleosides in length. In some embodiments, an oligonucleotide is 30 nucleosides in length. In some embodiments, an oligonucleotide is 31 nucleosides in length. In some embodiments, an oligonucleotide is 32 nucleosides in length. In some embodiments, an oligonucleotide is 33 nucleosides in length. In some embodiments, an oligonucleotide is 34 nucleosides in length. In some embodiments, an oligonucleotide is 35 nucleosides in length. In some embodiments, an oligonucleotide is 36 nucleosides in length. In some embodiments, an oligonucleotide is 37 nucleosides in length. In some embodiments, an oligonucleotide is 38 nucleosides in length. In some embodiments, an oligonucleotide is 39 nucleosides in length. In some embodiments, an oligonucleotide is 40 nucleosides in length. In some embodiments, each nucleoside counted in an oligonucleotide length independently comprises a nucleobase comprising a ring having at least one nitrogen ring atom. In some embodiments, each nucleoside counted in an oligonucleotide length independently comprises A, T, C, G, or U, or optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G or U.

[0080] Oligonucleotide type: As used herein, the phrase “oligonucleotide type” is used to define an oligonucleotide that has a particular base sequence, pattern of backbone linkages (i.e., pattern of internucleotidic linkage types, for example, phosphate, phosphorothioate, n001, etc.), pattern of backbone chiral centers (i.e., pattern of linkage phosphorus stereochemistry (Rp / Sp)), and pattern of backbone phosphorus modifications. In some embodiments, oligonucleotides of a common designated “type” are structurally identical to one another.

[0081] Optionally Substituted: As described herein, compounds of the disclosure may contain optionally substituted, substituted and / or unsubstituted moieties. In general, the term “substituted,” means that one or more hydrogens of the designated moiety are independently replaced with a substituent. Unless otherwise indicated, an “optionally substituted” group may independently have a substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with two or more substituents, the substituents may be either the same or different at every position. In some embodiments, an optionally substituted group is unsubstituted. In some embodiments, an optionally substituted group is substituted. Various substituents are described below.

[0082] Monovalent substituents are independently halogen; –(CH2)0–4R°; –(CH2)0–4OR°; −O(CH2)0-4Ro, – Page 25 of 274 12621671v1Attorney Docket No.: 2010581-1497 O–(CH2)0–4C(O)OR°; –(CH2)0–4CH(OR°)2; –(CH2)0–4Ph, which may be substituted with R°; −(CH2)0–4O(CH2)0–1Ph which may be substituted with R°; –CH=CHPh, which may be substituted with R°; –(CH2)0–4O(CH2)0–1- pyridyl which may be substituted with R°; –NO2; –CN; –N3; -(CH2)0–4N(R°)2; –(CH2)0–4N(R°)C(O)R°; – N(R°)C(S)R°; –(CH2)0–4N(R°)C(O)N(R°)2; −N(R°)C(S)N(R°)2; –(CH2)0–4N(R°)C(O)OR°; – N(R°)N(R°)C(O)R°; −N(R°)N(R°)C(O)N(R°)2; −N(R°)N(R°)C(O)OR°; –(CH2)0–4C(O)R°; –C(S)R°; –(CH2)0–4C(O)OR°; –(CH2)0–4C(O)SR°; −(CH2)0–4C(O)OSi(R°)3; –(CH2)0–4OC(O)R°; –OC(O)(CH2)0–4SR°, −SC(S)SR°; −(CH2)0–4SC(O)R°; –(CH2)0–4C(O)N(R°)2; –C(S)N(R°)2; –C(S)SR°; −SC(S)SR°, -(CH2)0–4OC(O)N(R°)2; -C(O)N(OR°)R°; –C(O)C(O)R°; –C(O)CH2C(O)R°; −C(NOR°)R°; -(CH2)0–4SSR°; –(CH2)0–4S(O)2R°; –(CH2)0–4S(O)2OR°; –(CH2)0–4OS(O)2R°; −S(O)2N(R°)2; -(CH2)0–4S(O)R°; –N(R°)S(O)2N(R°)2; – N(R°)S(O)2R°; –N(OR°)R°; −C(NH)N(R°)2; –Si(R°)3; –OSi(R°)3; −P(R°)2; −P(OR°)2; −OP(R°)2; −OP(OR°)2; −N(R°)P(R°)2; −B(R°)2; −OB(R°)2; −P(O)(R°)2; −OP(O)(R°)2; −N(R°)P(O)(R°)2; –(C1-4 straight or branched alkylene)O–N(R°)2; or –(C1-4straight or branched alkylene)C(O)O–N(R°)2; wherein each R° may be independently substituted as defined below and is independently hydrogen, C1-10 (e.g., C1-6, C1-5, C1-4, etc.) aliphatic, C1-10(e.g., C1-6, C1-5, C1-4, etc.) heteroaliphatic having 1-5 (e.g., 1, 2, 3, 4, 5, 1-5, 1-4, 1-2, etc.) heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, C6-10(e.g., C6, C10, etc.) aryl, 5-10 (e.g., 5-9, 5-6, 5, 6, 9, 10, etc.) membered heteroaryl having 1-4 (e.g., 1, 2, 3, 4, 1-4, 1-3, 1-2, etc.) heteroatoms independently selected from nitrogen, oxygen and sulfur, −CH2−(C6-10(e.g., C6, C10, etc.) aryl), −O(CH2)0-1(C6-10(e.g., C6, C10, etc.) aryl), −CH2−(5-10 (e.g., 5-9, 5-6, 5, 6, 9, 10, etc.) membered heteroaryl having 1-4 (e.g., 1, 2, 3, 4, 1-4, 1-3, 1-2, etc.) heteroatoms independently selected from nitrogen, oxygen and sulfur), −O(CH2)0-1(5-10 (e.g., 5-9, 5-6, 5, 6, 9, 10, etc.) membered heteroaryl having 1-4 (e.g., 1, 2, 3, 4, 1-4, 1-3, 1-2, etc.) heteroatoms independently selected from nitrogen, oxygen and sulfur), a 3-10 (e.g., 3-6, 5-6, 3, 4, 5, 6, 7, 8, 9, 10, etc.) membered, monocyclic, bicyclic, or polycyclic, saturated, or partially unsaturated ring having 0-5 (e.g., 0, 1, 2, 3, 4, 5, 1-5, 1-4, 1-2, etc.) heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3-10 (e.g., 3-6, 5-6, 3, 4, 5, 6, 7, 8, 9, 10, etc.) membered, monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated or aromatic ring (for aromatic ring, 5-10 (e.g., 5-9, 5-6, 5, 6, 9, 10, etc.) membered) having, in addition to the intervening atom(s), 0-5 (e.g., 0, 1, 2, 3, 4, 5, 1-5, 1-4, 1-2, etc.) heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, which may be substituted as defined below.

[0083] Monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, –(CH2)0–2R^, –(haloR^), –(CH2)0–2OH, – (CH2)0–2OR^, –(CH2)0–2CH(OR^)2; –O(haloR^), –CN, –N3, –(CH2)0–2C(O)R^, –(CH2)0–2C(O)OH, –(CH2)0–2C(O)OR^, –(CH2)0–2SR^, –(CH2)0–2SH, –(CH2)0–2NH2, –(CH2)0–2NHR^, –(CH2)0–2NR^2, –NO2, –SiR^3, – OSiR^3, -C(O)SR^,–(C1–4straight or branched alkylene)C(O)OR^, or –SSR^wherein each R^is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from Page 26 of 274 12621671v1Attorney Docket No.: 2010581-1497 C1–4aliphatic, –CH2Ph, –O(CH2)0–1Ph, or a 3-6 (e.g., 3, 4, 5, 6, 3-5, 5-6, etc.)-membered saturated, partially unsaturated, or aromatic ring (for aromatic ring, 5- or 6-membered) having 0-4 (e.g., 0, 1, 2, 3, 4, 1-4, 1-3, 1-2, etc.) heteroatoms independently selected from nitrogen, oxygen, and sulfur. Divalent substituents on a saturated carbon atom of R° are independently =O or =S.

[0084] Divalent substituents are independently the following: =O, =S, =NNR*2, =NNHC(O)R*, =NNHC(O)OR*, =NNHS(O)2R*, =NR*, =NOR*, −O(C(R*2))2–3O−, or −S(C(R*2))2–3S−, wherein each independent occurrence of R*is selected from hydrogen, C1-6aliphatic which may be substituted as defined below, or an unsubstituted 3-6 (e.g., 3, 4, 5, 6, 3-5, 5-6, etc.)-membered saturated, partially unsaturated, or aromatic ring (for aromatic ring, 5- or 6-membered) having 0-4 (e.g., 0, 1, 2, 3, 4, 1-4, 1-3, 1-2, etc.) heteroatoms independently selected from nitrogen, oxygen, and sulfur. Divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group are independently −O(CR*2)2–3O−, wherein each independent occurrence of R*is selected from hydrogen, C1-6aliphatic which may be substituted as defined below, or an unsubstituted 3-6 (e.g., 3, 4, 5, 6, 3-5, 5-6, etc.)-membered saturated, partially unsaturated, or aromatic ring (for aromatic ring, 5- or 6-membered) having 0-4 (e.g., 0, 1, 2, 3, 4, 1-4, 1-3, 1-2, etc.) heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0085] Substituents on the aliphatic group of R*are independently halogen, –R^, -(haloR^), –OH, −OR^, –O(haloR^), –CN, –C(O)OH, –C(O)OR^, –NH2, –NHR^, –NR^2, or –NO2, wherein each R^is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1–4 aliphatic, – CH2Ph, –O(CH2)0–1Ph, or a 3-6 (e.g., 3, 4, 5, 6, 3-5, 5-6, etc.)-membered saturated, partially unsaturated, or aromatic ring (for aromatic ring, 5- or 6-membered) having 0-4 (e.g., 0, 1, 2, 3, 4, 1-4, 1-3, 1-2, etc.) heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0086] Substituents on a substitutable nitrogen are independently –R†, −NR†2, −C(O)R†, –C(O)OR†, – C(O)C(O)R†, –C(O)CH2C(O)R†, –S(O)2R†, –S(O)2NR†2, −C(S)NR†2, –C(NH)NR†2, or –N(R†)S(O)2R†; wherein each R†is independently hydrogen, C1–6 aliphatic which may be substituted as defined below, unsubstituted – OPh, or an unsubstituted 3-6 (e.g., 3, 4, 5, 6, 3-5, 5-6, etc.)-membered saturated, partially unsaturated, or aromatic ring (for aromatic ring, 5- or 6-membered) having 0-4 (e.g., 0, 1, 2, 3, 4, 1-4, 1-3, 1-2, etc.) heteroatoms independently selected from nitrogen, oxygen, and sulfur, or, notwithstanding the definition above, two independent occurrences of R†, taken together with their intervening atom(s) form an unsubstituted 3-12 (e.g., 3-10, 3-6, 5-10, 5-6, 3, 4, 5, 6, 7, 8, 9, 10, etc.) membered saturated, partially unsaturated, or aryl mono– or bicyclic ring having 0-4 (e.g., 0, 1, 2, 3, 4, 1-4, 1-3, 1-2, etc.) heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0087] Substituents on the aliphatic group of R†are independently halogen, −R^, -(haloR^), −OH, –OR^, –O(haloR^), –CN, –C(O)OH, –C(O)OR^, –NH2, –NHR^, –NR^2, or –NO2, wherein each R^is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1–4 aliphatic, – CH2Ph, –O(CH2)0–1Ph, or a 3-6 (e.g., 3, 4, 5, 6, 3-5, 5-6, etc.)-membered saturated, partially unsaturated, or aromatic ring (for aromatic ring, 5- or 6-membered) having 0-4 (e.g., 0, 1, 2, 3, 4, 1-4, 1-3, 1-2, etc.) heteroatoms Page 27 of 274 12621671v1Attorney Docket No.: 2010581-1497 independently selected from nitrogen, oxygen, and sulfur.

[0088] Partially unsaturated: As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.

[0089] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, an active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0090] Pharmaceutically acceptable: As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0091] Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations. Page 28 of 274 12621671v1Attorney Docket No.: 2010581-1497

[0092] Pharmaceutically acceptable salt: The term “pharmaceutically acceptable salt”, as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, pharmaceutically acceptable salt include, but are not limited to, nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, 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, p-toluenesulfonate, undecanoate, valerate salts, and the like. In some embodiments, a provided compound comprises one or more acidic groups, e.g., an oligonucleotide, and a pharmaceutically acceptable salt is an alkali, alkaline earth metal, or ammonium (e.g., an ammonium salt of N(R)3, wherein each R is independently defined and described in the present disclosure) salt. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, a pharmaceutically acceptable salt is a sodium salt. In some embodiments, a pharmaceutically acceptable salt is a potassium salt. In some embodiments, a pharmaceutically acceptable salt is a calcium salt. In some embodiments, pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate. In some embodiments, a provided compound comprises more than one acid groups, for example, an oligonucleotide may comprise two or more acidic groups (e.g., in natural phosphate linkages and / or modified internucleotidic linkages). In some embodiments, a pharmaceutically acceptable salt, or generally a salt, of such a compound comprises two or more cations, which can be the same or different. In some embodiments, in a pharmaceutically acceptable salt (or generally, a salt), all ionizable hydrogen (e.g., in an aqueous solution with a pKa no more than about 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2; in some embodiments, no more than about 7; in some embodiments, no more than about 6; in some embodiments, no more than about 5; in some embodiments, no more than about 4; in some embodiments, no more than about 3) in the acidic groups are replaced with cations. In some embodiments, each phosphorothioate and phosphate group independently exists in its salt form (e.g., if sodium salt, Page 29 of 274 12621671v1Attorney Docket No.: 2010581-1497 −O−P(O)(SNa)−O− and −O−P(O)(ONa)−O−, respectively). In some embodiments, each phosphorothioate and phosphate internucleotidic linkage independently exists in its salt form (e.g., if sodium salt, −O−P(O)(SNa)−O− and −O−P(O)(ONa)−O−, respectively). In some embodiments, a pharmaceutically acceptable salt is a sodium salt of an oligonucleotide. In some embodiments, a pharmaceutically acceptable salt is a sodium salt of an oligonucleotide, wherein each acidic phosphate and modified phosphate group (e.g., phosphorothioate, phosphate, etc.), if any, exists as a salt form (all sodium salt).

[0093] Protecting group: The term “protecting group,” as used herein, is well known in the art and includes those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rdedition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Also included are those protecting groups specially adapted for nucleoside and nucleotide chemistry described in Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al. 06 / 2012, the entirety of Chapter 2 is incorporated herein by reference. Suitable amino–protecting groups include methyl carbamate, ethyl carbamante, 9–fluorenylmethyl carbamate (Fmoc), 9–(2–sulfo)fluorenylmethyl carbamate, 9–(2,7– dibromo)fluoroenylmethyl carbamate, 2,7–di–t–butyl–[9–(10,10–dioxo–10,10,10,10– tetrahydrothioxanthyl)]methyl carbamate (DBD–Tmoc), 4–methoxyphenacyl carbamate (Phenoc), 2,2,2– trichloroethyl carbamate (Troc), 2–trimethylsilylethyl carbamate (Teoc), 2–phenylethyl carbamate (hZ), 1–(1– adamantyl)–1–methylethyl carbamate (Adpoc), 1,1–dimethyl–2–haloethyl carbamate, 1,1–dimethyl–2,2– dibromoethyl carbamate (DB–t–BOC), 1,1–dimethyl–2,2,2–trichloroethyl carbamate (TCBOC), 1–methyl–1– (4–biphenylyl)ethyl carbamate (Bpoc), 1–(3,5–di–t–butylphenyl)–1–methylethyl carbamate (t–Bumeoc), 2– (2’– and 4’–pyridyl)ethyl carbamate (Pyoc), 2–(N,N–dicyclohexylcarboxamido)ethyl carbamate, t–butyl carbamate (BOC), 1–adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1– isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4–nitrocinnamyl carbamate (Noc), 8–quinolyl carbamate, N–hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p– methoxybenzyl carbamate (Moz), p–nitobenzyl carbamate, p–bromobenzyl carbamate, p–chlorobenzyl carbamate, 2,4–dichlorobenzyl carbamate, 4–methylsulfinylbenzyl carbamate (Msz), 9–anthrylmethyl carbamate, diphenylmethyl carbamate, 2–methylthioethyl carbamate, 2–methylsulfonylethyl carbamate, 2–(p– toluenesulfonyl)ethyl carbamate, [2–(1,3–dithianyl)]methyl carbamate (Dmoc), 4–methylthiophenyl carbamate (Mtpc), 2,4–dimethylthiophenyl carbamate (Bmpc), 2–phosphonioethyl carbamate (Peoc), 2– triphenylphosphonioisopropyl carbamate (Ppoc), 1,1–dimethyl–2–cyanoethyl carbamate, m–chloro–p– acyloxybenzyl carbamate, p–(dihydroxyboryl)benzyl carbamate, 5–benzisoxazolylmethyl carbamate, 2– (trifluoromethyl)–6–chromonylmethyl carbamate (Tcroc), m–nitrophenyl carbamate, 3,5–dimethoxybenzyl carbamate, o–nitrobenzyl carbamate, 3,4–dimethoxy–6–nitrobenzyl carbamate, phenyl(o–nitrophenyl)methyl carbamate, phenothiazinyl–(10)–carbonyl derivative, N’–p–toluenesulfonylaminocarbonyl derivative, N’– phenylaminothiocarbonyl derivative, t–amyl carbamate, S–benzyl thiocarbamate, p–cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p– decyloxybenzyl carbamate, 2,2–dimethoxycarbonylvinyl carbamate, o–(N,N–dimethylcarboxamido)benzyl Page 30 of 274 12621671v1Attorney Docket No.: 2010581-1497 carbamate, 1,1–dimethyl–3–(N,N–dimethylcarboxamido)propyl carbamate, 1,1–dimethylpropynyl carbamate, di(2–pyridyl)methyl carbamate, 2–furanylmethyl carbamate, 2–iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p–(p’–methoxyphenylazo)benzyl carbamate, 1–methylcyclobutyl carbamate, 1–methylcyclohexyl carbamate, 1–methyl–1–cyclopropylmethyl carbamate, 1–methyl–1–(3,5– dimethoxyphenyl)ethyl carbamate, 1–methyl–1–(p–phenylazophenyl)ethyl carbamate, 1–methyl–1– phenylethyl carbamate, 1–methyl–1–(4–pyridyl)ethyl carbamate, phenyl carbamate, p–(phenylazo)benzyl carbamate, 2,4,6–tri–t–butylphenyl carbamate, 4–(trimethylammonium)benzyl carbamate, 2,4,6– trimethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3–phenylpropanamide, picolinamide, 3–pyridylcarboxamide, N–benzoylphenylalanyl derivative, benzamide, p–phenylbenzamide, o–nitophenylacetamide, o–nitrophenoxyacetamide, acetoacetamide, (N’–dithiobenzyloxycarbonylamino)acetamide, 3–(p–hydroxyphenyl)propanamide, 3–(o– nitrophenyl)propanamide, 2–methyl–2–(o–nitrophenoxy)propanamide, 2–methyl–2–(o– phenylazophenoxy)propanamide, 4–chlorobutanamide, 3–methyl–3–nitrobutanamide, o–nitrocinnamide, N– acetylmethionine derivative, o–nitrobenzamide, o–(benzoyloxymethyl)benzamide, 4,5–diphenyl–3–oxazolin– 2–one, N–phthalimide, N–dithiasuccinimide (Dts), N–2,3–diphenylmaleimide, N–2,5–dimethylpyrrole, N– 1,1,4,4–tetramethyldisilylazacyclopentane adduct (STABASE), 5–substituted 1,3–dimethyl–1,3,5– triazacyclohexan–2–one, 5–substituted 1,3–dibenzyl–1,3,5–triazacyclohexan–2–one, 1–substituted 3,5– dinitro–4–pyridone, N–methylamine, N–allylamine, N–[2–(trimethylsilyl)ethoxy]methylamine (SEM), N–3– acetoxypropylamine, N–(1–isopropyl–4–nitro–2–oxo–3–pyroolin–3–yl)amine, quaternary ammonium salts, N–benzylamine, N–di(4–methoxyphenyl)methylamine, N–5–dibenzosuberylamine, N–triphenylmethylamine (Tr), N–[(4–methoxyphenyl)diphenylmethyl]amine (MMTr), N–9–phenylfluorenylamine (PhF), N–2,7– dichloro–9–fluorenylmethyleneamine, N–ferrocenylmethylamino (Fcm), N–2–picolylamino N’–oxide, N–1,1– dimethylthiomethyleneamine, N–benzylideneamine, N–p–methoxybenzylideneamine, N– diphenylmethyleneamine, N–[(2–pyridyl)mesityl]methyleneamine, N–(N’,N’– dimethylaminomethylene)amine, N,N’–isopropylidenediamine, N–p–nitrobenzylideneamine, N– salicylideneamine, N–5–chlorosalicylideneamine, N–(5–chloro–2–hydroxyphenyl)phenylmethyleneamine, N– cyclohexylideneamine, N–(5,5–dimethyl–3–oxo–1–cyclohexenyl)amine, N–borane derivative, N– diphenylborinic acid derivative, N–[phenyl(pentacarbonylchromium– or tungsten)carbonyl]amine, N–copper chelate, N–zinc chelate, N–nitroamine, N–nitrosoamine, amine N–oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o–nitrobenzenesulfenamide (Nps), 2,4– dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2–nitro–4–methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3–nitropyridinesulfenamide (Npys), p–toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,–trimethyl–4–methoxybenzenesulfonamide (Mtr), 2,4,6– trimethoxybenzenesulfonamide (Mtb), 2,6–dimethyl–4–methoxybenzenesulfonamide (Pme), 2,3,5,6– tetramethyl–4–methoxybenzenesulfonamide (Mte), 4–methoxybenzenesulfonamide (Mbs), 2,4,6– Page 31 of 274 12621671v1Attorney Docket No.: 2010581-1497 trimethylbenzenesulfonamide (Mts), 2,6–dimethoxy–4–methylbenzenesulfonamide (iMds), 2,2,5,7,8– pentamethylchroman–6–sulfonamide (Pmc), methanesulfonamide (Ms), β–trimethylsilylethanesulfonamide (SES), 9–anthracenesulfonamide, 4–(4’,8’–dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

[0094] Suitably protected carboxylic acids further include, but are not limited to, silyl–, alkyl–, alkenyl–, aryl–, and arylalkyl–protected carboxylic acids. Examples of suitable silyl groups include trimethylsilyl, triethylsilyl, t–butyldimethylsilyl, t–butyldiphenylsilyl, triisopropylsilyl, and the like. Examples of suitable alkyl groups include methyl, benzyl, p–methoxybenzyl, 3,4–dimethoxybenzyl, trityl, t–butyl, tetrahydropyran– 2–yl. Examples of suitable alkenyl groups include allyl. Examples of suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl. Examples of suitable arylalkyl groups include optionally substituted benzyl (e.g., p–methoxybenzyl (MPM), 3,4–dimethoxybenzyl, O–nitrobenzyl, p–nitrobenzyl, p–halobenzyl, 2,6–dichlorobenzyl, p–cyanobenzyl), and 2– and 4–picolyl.

[0095] Suitable hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t–butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p– methoxybenzyloxymethyl (PMBM), (4–methoxyphenoxy)methyl (p–AOM), guaiacolmethyl (GUM), t– butoxymethyl, 4–pentenyloxymethyl (POM), siloxymethyl, 2–methoxyethoxymethyl (MEM), 2,2,2– trichloroethoxymethyl, bis(2–chloroethoxy)methyl, 2–(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3–bromotetrahydropyranyl, tetrahydrothiopyranyl, 1–methoxycyclohexyl, 4– methoxytetrahydropyranyl (MTHP), 4–methoxytetrahydrothiopyranyl, 4–methoxytetrahydrothiopyranyl S,S– dioxide, 1–[(2–chloro–4–methyl)phenyl]–4–methoxypiperidin–4–yl (CTMP), 1,4–dioxan–2–yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a–octahydro–7,8,8–trimethyl–4,7– methanobenzofuran–2–yl, 1–ethoxyethyl, 1–(2–chloroethoxy)ethyl, 1–methyl–1–methoxyethyl, 1–methyl–1– benzyloxyethyl, 1–methyl–1–benzyloxy–2–fluoroethyl, 2,2,2–trichloroethyl, 2–trimethylsilylethyl, 2– (phenylselenyl)ethyl, t–butyl, allyl, p–chlorophenyl, p–methoxyphenyl, 2,4–dinitrophenyl, benzyl, p– methoxybenzyl, 3,4–dimethoxybenzyl, o–nitrobenzyl, p–nitrobenzyl, p–halobenzyl, 2,6–dichlorobenzyl, p– cyanobenzyl, p–phenylbenzyl, 2–picolyl, 4–picolyl, 3–methyl–2–picolyl N–oxido, diphenylmethyl, p,p’– dinitrobenzhydryl, 5–dibenzosuberyl, triphenylmethyl, α–naphthyldiphenylmethyl, p– methoxyphenyldiphenylmethyl, di(p–methoxyphenyl)phenylmethyl, tri(p–methoxyphenyl)methyl, 4–(4’– bromophenacyloxyphenyl)diphenylmethyl, 4,4’,4’’–tris(4,5–dichlorophthalimidophenyl)methyl, 4,4’,4’’– tris(levulinoyloxyphenyl)methyl, 4,4’,4’’–tris(benzoyloxyphenyl)methyl, 3–(imidazol–1–yl)bis(4’,4’’– dimethoxyphenyl)methyl, 1,1–bis(4–methoxyphenyl)–1’–pyrenylmethyl, 9–anthryl, 9–(9–phenyl)xanthenyl, 9–(9–phenyl–10–oxo)anthryl, 1,3–benzodithiolan–2–yl, benzisothiazolyl S,S–dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t–butyldimethylsilyl (TBDMS), t–butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri–p– xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t–butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, Page 32 of 274 12621671v1Attorney Docket No.: 2010581-1497 triphenylmethoxyacetate, phenoxyacetate, p–chlorophenoxyacetate, 3–phenylpropionate, 4–oxopentanoate (levulinate), 4,4–(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4– methoxycrotonate, benzoate, p–phenylbenzoate, 2,4,6–trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9–fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2–trichloroethyl carbonate (Troc), 2– (trimethylsilyl)ethyl carbonate (TMSEC), 2–(phenylsulfonyl) ethyl carbonate (Psec), 2–(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p–nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p–methoxybenzyl carbonate, alkyl 3,4–dimethoxybenzyl carbonate, alkyl o–nitrobenzyl carbonate, alkyl p–nitrobenzyl carbonate, alkyl S–benzyl thiocarbonate, 4–ethoxy–1– napththyl carbonate, methyl dithiocarbonate, 2–iodobenzoate, 4–azidobutyrate, 4–nitro–4–methylpentanoate, o–(dibromomethyl)benzoate, 2–formylbenzenesulfonate, 2–(methylthiomethoxy)ethyl, 4– (methylthiomethoxy)butyrate, 2–(methylthiomethoxymethyl)benzoate, 2,6–dichloro–4– methylphenoxyacetate, 2,6–dichloro–4–(1,1,3,3–tetramethylbutyl)phenoxyacetate, 2,4–bis(1,1– dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)–2–methyl–2– butenoate, o–(methoxycarbonyl)benzoate, α–naphthoate, nitrate, alkyl N,N,N’,N’– tetramethylphosphorodiamidate, alkyl N–phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4– dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). For protecting 1,2– or 1,3–diols, the protecting groups include methylene acetal, ethylidene acetal, 1–t–butylethylidene ketal, 1–phenylethylidene ketal, (4–methoxyphenyl)ethylidene acetal, 2,2,2–trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p– methoxybenzylidene acetal, 2,4–dimethoxybenzylidene ketal, 3,4–dimethoxybenzylidene acetal, 2– nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1–methoxyethylidene ortho ester, 1–ethoxyethylidine ortho ester, 1,2–dimethoxyethylidene ortho ester, α– methoxybenzylidene ortho ester, 1–(N,N–dimethylamino)ethylidene derivative, α–(N,N’– dimethylamino)benzylidene derivative, 2–oxacyclopentylidene ortho ester, di–t–butylsilylene group (DTBS), 1,3–(1,1,3,3–tetraisopropyldisiloxanylidene) derivative (TIPDS), tetra–t–butoxydisiloxane–1,3–diylidene derivative (TBDS), cyclic carbonates, cyclic boronates, ethyl boronate, and phenyl boronate.

[0096] In some embodiments, a hydroxyl protecting group is acetyl, t-butyl, tbutoxymethyl, methoxymethyl, tetrahydropyranyl, 1 -ethoxyethyl, 1 -(2-chloroethoxy)ethyl, 2- trimethylsilylethyl, p- chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6- dichlorobenzyl, diphenylmethyl, p- nitrobenzyl, triphenylmethyl (trityl), 4,4'-dimethoxytrityl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t- butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifiuoroacetyl, pivaloyl, 9- fluorenylmethyl carbonate, mesylate, tosylate, triflate, trityl, monomethoxytrityl (MMTr), 4,4'-dimethoxytrityl, (DMTr) and 4,4',4''-trimethoxytrityl (TMTr), 2-cyanoethyl (CE or Cne), 2- (trimethylsilyl)ethyl (TSE), 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl 2-(4-nitrophenyl)ethyl (NPE), 2- (4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl, 2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6- trimethylphenyl, 2-(2-nitrophenyl)ethyl, butylthiocarbonyl, 4,4',4''-tris(benzoyloxy)trityl, diphenylcarbamoyl, Page 33 of 274 12621671v1Attorney Docket No.: 2010581-1497 levulinyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9- phenylxanthen-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthine-9-y1 (MOX). In some embodiments, each of the hydroxyl protecting groups is, independently selected from acetyl, benzyl, t- butyldimethylsilyl, t- butyldiphenylsilyl and 4,4'-dimethoxytrityl. In some embodiments, the hydroxyl protecting group is selected from the group consisting of trityl, monomethoxytrityl and 4,4'-dimethoxytrityl group. In some embodiments, a phosphorous linkage protecting group is a group attached to the phosphorous linkage (e.g., an internucleotidic linkage) throughout oligonucleotide synthesis. In some embodiments, a protecting group is attached to a sulfur atom of an phosphorothioate group. In some embodiments, a protecting group is attached to an oxygen atom of an internucleotide phosphorothioate linkage. In some embodiments, a protecting group is attached to an oxygen atom of the internucleotide phosphate linkage. In some embodiments a protecting group is 2-cyanoethyl (CE or Cne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl, o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe), 2-phenylethyl, 3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl, 4-methylthio-l-butyl, 2- cyano-1,1-dimethylethyl, 4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2- pyridyl)]aminoethyl, 2-(N-formyl,N-methyl)aminoethyl, or 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl.

[0097] Sample: As used herein, the term “sample” typically refers to an aliquot of material obtained or derived from a source of interest. In some embodiments, a source of interest is a biological or environmental source. In some embodiments, a source of interest may be or comprise a cell or an organism, such as a microbe, a plant, or an animal (e.g., a human). In some embodiments, a source of interest is or comprises biological tissue or fluid. In some embodiments, a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secreations, vitreous humour, vomit, and / or combinations or component(s) thereof. In some embodiments, a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and / or a transcellular fluid. In some embodiments, a biological fluid may be or comprise a plant exudate. In some embodiments, a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., brocheoalvealar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and / or purification of certain components, etc. Page 34 of 274 12621671v1Attorney Docket No.: 2010581-1497

[0098] Subject: As used herein, the term “subject” or “test subject” refers to any organism to which a compound (e.g., an oligonucleotide) or composition is administered in accordance with the present disclosure e.g., for experimental, diagnostic, prophylactic and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, a subject is a human. In some embodiments, a subject may be suffering from and / or susceptible to a disease, disorder and / or condition.

[0099] Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. A base sequence which is substantially identical or complementary to a second sequence is not fully identical or complementary to the second sequence, but is mostly or nearly identical or complementary to the second sequence. In some embodiments, an oligonucleotide with a substantially complementary sequence to another oligonucleotide or nucleic acid forms duplex with the oligonucleotide or nucleic acid in a similar fashion as an oligonucleotide with a fully complementary sequence. In addition, one of ordinary skill in the biological and / or chemical arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and / or chemical phenomena.

[0100] Sugar: The term “sugar” refers to a monosaccharide or polysaccharide in closed and / or open form. In some embodiments, sugars are monosaccharides. In some embodiments, sugars are polysaccharides. Sugars include ribose, deoxyribose, pentofuranose, pentopyranose, and hexopyranose moieties. As used herein, the term “sugar” also encompasses structural analogs used in lieu of conventional sugar molecules, such as glycol, polymer of which forms the backbone of the nucleic acid analog, glycol nucleic acid (“GNA”), etc. As used herein, the term “sugar” also encompasses structural analogs used in lieu of natural or naturally-occurring nucleotides, such as modified sugars and nucleotide sugars. In some embodiments, a sugar is a RNA or DNA sugar (ribose or deoxyribose). In some embodiments, a sugar is a modified ribose or deoxyribose sugar, e.g., 2’-modified, 5’-modified, etc. As described herein, in some embodiments, when used in oligonucleotides and / or nucleic acids, modified sugars may provide one or more desired properties, activities, etc. In some embodiments, a sugar is optionally substituted ribose or deoxyribose. In some embodiments, a “sugar” refers to a sugar unit in an oligonucleotide or a nucleic acid.

[0101] Susceptible to: An individual who is “susceptible to” a disease, disorder and / or condition is one who has a higher risk of developing the disease, disorder and / or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition is predisposed to have that disease, disorder and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition may not have been diagnosed with the disease, disorder and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition may exhibit symptoms of the disease, disorder and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition may not exhibit symptoms of the disease, disorder and / or Page 35 of 274 12621671v1Attorney Docket No.: 2010581-1497 condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0102] Therapeutic agent: As used herein, the term “therapeutic agent” in general refers to any agent that elicits a desired effect (e.g., a desired biological, clinical, or pharmacological effect) when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, an appropriate population is a population of subjects suffering from and / or susceptible to a disease, disorder or condition. In some embodiments, an appropriate population is a population of model organisms. In some embodiments, an appropriate population may be defined by one or more criterion such as age group, gender, genetic background, preexisting clinical conditions, prior exposure to therapy. In some embodiments, a therapeutic agent is a substance that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms or features of a disease, disorder, and / or condition in a subject when administered to the subject in an effective amount. In some embodiments, a “therapeutic agent” is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a “therapeutic agent” is an agent for which a medical prescription is required for administration to humans. In some embodiments, a therapeutic agent is a provided compound, e.g., a provided oligonucleotide.

[0103] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and / or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and / or reduces incidence of one or more symptoms or features of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0104] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, Page 36 of 274 12621671v1Attorney Docket No.: 2010581-1497 and / or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0105] Unsaturated: The term "unsaturated," as used herein, means that a moiety has one or more units of unsaturation.

[0106] Wild-type: As used herein, the term “wild-type” has its art-understood meaning that refers to an entity having a structure and / or activity as found in nature in a “normal” (as contrasted with mutant, diseased, altered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0107] As those skilled in the art will appreciate, methods and compositions described herein relating to provided compounds (e.g., oligonucleotides) generally also apply to pharmaceutically acceptable salts of such compounds. Description of Certain Embodiments

[0108] Oligonucleotides are useful tools for a wide variety of applications. For example, oligonucleotides are useful in therapeutic, diagnostic, and research applications. The use of unmodified DNA or RNA is limited, for example, by their susceptibility to endo- and exo-nucleases. As such, various synthetic counterparts have been developed to circumvent these shortcomings and / or to further improve various properties and activities. These include synthetic oligonucleotides that contain chemical modifications, e.g., base modifications, sugar modifications, backbone modifications, etc., which, among other things, render these molecules less susceptible to degradation and improve other properties and / or activities. Among many others, one modification is PN internucleotidic linkage which can be utilized to improve oligonucleotide properties and activities. From a structural point of view, modifications to internucleotidic linkages can introduce chirality, and certain properties may be affected by configurations of linkage phosphorus atoms of modified internucleotidic linkages such as PN internucleotidic linkages. Among other things, the present disclosure demonstrates that positioning and stereochemistry of PN internucleotidic linkages can impact various properties and activities of oligonucleotides. In some embodiments, the present disclosure identifies positions and stereochemistry for PN incorporation, e.g., into middle portions of oligonucleotides (e.g., core regions, L-8to L3, etc.).

[0109] In some embodiments, provided technologies comprise control of chiral linkage phosphorus, e.g., in chiral PO, PS and / or PN linkages. In some embodiments, internucleotidic linkages of an oligonucleotide comprise or consist of about 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-40, 1-50, or about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more chirally controlled internucleotidic linkages. In some embodiments, the present disclosure provides an oligonucleotide composition comprising oligonucleotides that comprise at least one chirally controlled internucleotidic linkage. In some embodiments, there are at least 5 chirally controlled internucleotidic linkages. In some embodiments, there are at least 10 chirally controlled internucleotidic linkages. In some embodiments, the present disclosure provides a stereorandom oligonucleotide composition. In some embodiments, each PS internucleotidic linkage is Page 37 of 274 12621671v1Attorney Docket No.: 2010581-1497 independently chirally controlled. In some embodiments, each phosphorothioate internucleotidic linkage is independently chirally controlled. In some embodiments, each PN internucleotidic linkage is independently chirally controlled. In some embodiments, each n001 linkage is independently chirally controlled. In some embodiments, each n006 linkage is independently chirally controlled. In some embodiments, each chiral internucleotidic linkage is independently chirally controlled.

[0110] In some embodiments, an oligonucleotide comprises one or more (e.g., about 1-5, 1-10, 1-15, 1- 20, 1-25, 1-30, 1-40, 1-50, or about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) negatively charged internucleotidic linkages (e.g., phosphorothioate internucleotidic linkages, natural phosphate linkages, etc.). In some embodiments, an oligonucleotide comprises one or more (e.g., about 1-5, 1- 10, 1-15, 1-20, 1-25, 1-30, 1-40, 1-50, or about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) internucleotidic linkages each of which is independently a PS linkage (e.g., a phosphorothioate internucleotidic linkage). In some embodiments, an oligonucleotide comprises one or more (e.g., about 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-40, 1-50, or about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) internucleotidic linkages each of which is independently a PN linkage (e.g., n001). In some embodiments, one or more (e.g., about 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-40, 1-50, or about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) internucleotidic linkages are each independently a non-negatively charged internucleotidic linkage. In some embodiments, one or more (e.g., about 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-40, 1-50, or about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) internucleotidic linkages are each independently a neutral internucleotidic linkage (e.g., n001). In some embodiments, a non-negatively charged internucleotidic linkage is a phosphoryl guanidine internucleotidic linkage (e.g., n001). In some embodiments, the number of PS internucleotidic linkages in an oligonucleotide is about or at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, each PS internucleotidic linkage is independently a phosphorothioate internucleotidic linkage. In some embodiments, the number of PN internucleotidic linkages in an oligonucleotide is independently about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. In some embodiments, a PN internucleotidic linkage is a phosphoramidate internucleotidic linkage. In some embodiments, a PN internucleotidic linkage is a phosphoryl guanidine internucleotidic linkage. In some embodiments, a PN internucleotidic linkage is a n001 internucleotidic linkage. In some embodiments, a PN internucleotidic linkage is a n006 internucleotidic linkage. In some embodiments, a PN internucleotidic linkage is a MsPA internucleotidic linkage. In some embodiments, each PN internucleotidic linkage is independently a n001 internucleotidic linkage. In some embodiments, each PN internucleotidic linkage is independently a n006 internucleotidic linkage. In some embodiments, each PN internucleotidic linkage is independently a MsPA internucleotidic linkage. In some embodiments, a PN internucleotidic linkage is Rp. In some embodiments, a PN internucleotidic linkage is Sp. In some embodiments, a PN internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, a PN internucleotidic linkage is a positively charged internucleotidic linkage. In some embodiments, a PN internucleotidic linkage is a negatively charged Page 38 of 274 12621671v1Attorney Docket No.: 2010581-1497 internucleotidic linkage. Among other things, the present disclosure provides methods for using PN internucleotidic linkages to provide oligonucleotides with improved properties and / or activities. Oligonucleotides

[0111] Among other things, the present disclosure provides oligonucleotides of various designs, which may comprise various nucleobases and patterns thereof, various sugars and patterns thereof, various internucleotidic linkages and patterns thereof, and / or additional chemical moieties and patterns thereof as described in the present disclosure. Certain examples of various modifications and patterns thereof are described below.

[0112] In some embodiments, provided technologies (e.g., oligonucleotides, compositions, methods, etc.) are useful for modulate levels of target nucleic acids (e.g., transcripts) and products encoded thereby (e.g., polypeptides). As described herein, oligonucleotides and compositions of the present disclosure are useful for many applications. For example, in some embodiments, they are useful for preventing or treating various conditions, diseases or disorders.

[0113] In some embodiments, an oligonucleotide comprises a structural element or a portion thereof described herein, e.g., in a Table. In some embodiments, an oligonucleotide comprises a base sequence (or a portion thereof) described herein, wherein each T can be independently substituted with U and vice versa, a chemical modification or a pattern of chemical modifications (or a portion thereof), and / or a format or a portion thereof described herein.

[0114] In some embodiments, oligonucleotides are manufactured stereoselectively, e.g., with respect to one or more or all chiral linkage phosphorus. In some embodiments, oligonucleotides are provided as chirally controlled oligonucleotide compositions. In some embodiments, oligonucleotides are manufactured stereorandomly without pre-determined stereochemical control. Among other things, the present disclosure provides technologies for preparing chirally controlled oligonucleotides. In some embodiments, oligonucleotides of the present disclosure are about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%- 100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, pure. In some embodiments, purity is assessed by suitable separation (e.g., various LC technologies such as HPLC, UPLC, etc.) and detection (e.g., UV at a suitable wavelength such as about 254 nm). In some embodiments, purity is peak area %. Various technologies are available for assessing oligonucleotide purity and can be utilized in accordance with the present disclosure. In some embodiments, internucleotidic linkages of oligonucleotides comprise or consist of one or more (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 5-50, 5-40, 5-30, 5-25, 5-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) chiral linkage phosphorus, each of which independently has a diastereopurity of about or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, typically about or at Page 39 of 274 12621671v1Attorney Docket No.: 2010581-1497 least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%. In some embodiments, de of one or more (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 5-50, 5-40, 5-30, 5-25, 5-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) or each chiral linkage phosphorus is independently about or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, typically about or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%. In some embodiments, oligonucleotides of the present disclosure have a diastereopurity of about or at least about (DS)CIL, wherein DS is a diastereopurity as described in the present disclosure or is about or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% or more) and CIL is the number of chiral linkage phosphorus (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 5-50, 5-40, 5-30, 5-25, 5-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more). In some embodiments, DS is about 95%-100%.

[0115] In some embodiments, oligonucleotides contain increased levels of one or more isotopes. In some embodiments, oligonucleotides are labeled, e.g., by one or more isotopes of one or more elements, e.g., hydrogen, carbon, nitrogen, etc. In some embodiments, oligonucleotides in provided compositions, e.g., oligonucleotides of a plurality of a composition, comprise base modifications, sugar modifications, and / or internucleotidic linkage modifications, wherein the oligonucleotides contain an enriched level of deuterium. In some embodiments, oligonucleotides are labeled with deuterium (replacing −1H with −2H) at one or more positions. In some embodiments, one or more1H of an oligonucleotide chain or any moiety conjugated to the oligonucleotide chain (e.g., a targeting moiety, etc.) is substituted with2H. Such oligonucleotides can be used in compositions and methods described herein.

[0116] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides which: 1) share a common base sequence; and 2) comprise one or more modified sugar moieties and / or modified internucleotidic linkages.

[0117] In some embodiments, oligonucleotides having a common base sequence may have the same pattern of nucleoside modifications, e.g., sugar modifications, base modifications, etc. In some embodiments, a pattern of nucleoside modifications may be represented by a combination of locations and modifications. In some embodiments, a pattern of backbone linkages comprises locations and types (e.g., phosphate, phosphorothioate, substituted phosphorothioate, etc.) of each internucleotidic linkage.

[0118] In some embodiments, oligonucleotides of a plurality, e.g., in provided compositions, share a common pattern of sugar modifications. In some embodiments, they share a common pattern of base modifications. In some embodiments, they share a common pattern of nucleoside modifications. In some embodiments, oligonucleotides of a plurality have the same constitution. In some embodiments, oligonucleotides of a plurality are identical.

[0119] As used in the present disclosure, in some embodiments, “one or more” is about 1-200, 1-150, 1- 100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, or about 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. In some embodiments, “one or more” is one. In some embodiments, “one or Page 40 of 274 12621671v1Attorney Docket No.: 2010581-1497 more” is two. In some embodiments, “one or more” is three. In some embodiments, “one or more” is four. In some embodiments, “one or more” is five. In some embodiments, “one or more” is six. In some embodiments, “one or more” is seven. In some embodiments, “one or more” is eight. In some embodiments, “one or more” is nine. In some embodiments, “one or more” is ten. In some embodiments, “one or more” is at least one. In some embodiments, “one or more” is at least two. In some embodiments, “one or more” is at least three. In some embodiments, “one or more” is at least four. In some embodiments, “one or more” is at least five. In some embodiments, “one or more” is at least six. In some embodiments, “one or more” is at least seven. In some embodiments, “one or more” is at least eight. In some embodiments, “one or more” is at least nine. In some embodiments, “one or more” is at least ten.

[0120] As used in the present disclosure, in some embodiments, “at least one” is about or at least about 1- 200, 1-150, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, or about or at least about 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. In some embodiments, “at least one” is one. In some embodiments, “at least one” is two. In some embodiments, “at least one” is three. In some embodiments, “at least one” is four. In some embodiments, “at least one” is five. In some embodiments, “at least one” is six. In some embodiments, “at least one” is seven. In some embodiments, “at least one” is eight. In some embodiments, “at least one” is nine. In some embodiments, “at least one” is ten.

[0121] In some embodiments, oligonucleotides are provided as salt forms. In some embodiments, oligonucleotides are provided as salts comprising negatively charged internucleotidic linkages (e.g., phosphorothioate internucleotidic linkages, natural phosphate linkages, etc.) existing as their salt forms. In some embodiments, oligonucleotides are provided as pharmaceutically acceptable salts. In some embodiments, oligonucleotides are provided as metal salts. In some embodiments, oligonucleotides are provided as sodium salts. In some embodiments, oligonucleotides are provided as metal salts, e.g., sodium salts, wherein each negatively charged internucleotidic linkage is independently in a salt form (e.g., for sodium salts, −O−P(O)(SNa)−O− for a phosphorothioate internucleotidic linkage, −O−P(O)(ONa)−O− for a natural phosphate linkage, etc.). In some embodiments, in a composition (e.g., in a buffered liquid composition) oligonucleotides may exist as multiple forms including multiple salt forms. Base Sequences

[0122] Oligonucleotides of the present disclosure comprise various nucleobases as described herein (e.g., A, T, C, G, U, etc.). In some embodiments, base sequences of oligonucleotides, as appreciated by those skilled in the art, have sufficient length and complementarity to their targets, e.g., RNA transcripts (e.g., pre-mRNA, mature mRNA, etc.) to modulate levels and / or activities of their targets, e.g., target-specific knockdown. In some embodiments, base sequence of an oligonucleotide has a sufficient length and complementarity to a transcript target to mediate target-specific knockdown. In some embodiments, a base sequence is complementary to that of a target or a portion thereof. In some embodiments, an oligonucleotide hybridizes to a target or a portion thereof, e.g., through base pairing. In some embodiments, an oligonucleotide selectively Page 41 of 274 12621671v1Attorney Docket No.: 2010581-1497 hybridizes to a target or a portion thereof through base pairing. In some embodiments, when hybridizing to a target or a portion thereof, each nucleobase of an oligonucleotide base-pairs with a nucleobase in a target. In some embodiments, there are one or more mismatches.

[0123] In some embodiments, base sequence of an oligonucleotide is or comprises a base sequence described herein (e.g., of an oligonucleotide in a Table) or a portion (e.g., a span of about 5-50, 5-40, 5-30, 5- 20, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or at least about 10, at least about 15, contiguous nucleobases) thereof with 0-5 (e.g., 0, 1, 2, 3, 4 or 5) mismatches, wherein each T can be independently substituted with U and vice versa. In some embodiments, base sequence of an oligonucleotide is or comprises a base sequence that is complementary to a complementary sequence of a base sequence described herein or a portion thereof (e.g., a span of about 5-50, 5-40, 5-30, 5-20, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or at least about 10, at least about 15, contiguous nucleobases) thereof with 0-5 (e.g., 0, 1, 2, 3, 4 or 5) mismatches, wherein each T can be independently substituted with U and vice versa. In some embodiments, an oligonucleotide hybridizes to a sequence that is or comprises a base sequence described herein (e.g., of an oligonucleotide in a Table) or a portion (e.g., a span of about 5-50, 5-40, 5-30, 5- 20, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or at least about 10, at least about 15, contiguous nucleobases) thereof with 0-5 (e.g., 0, 1, 2, 3, 4 or 5) mismatches, wherein each T can be independently substituted with U and vice versa. In some embodiments, an oligonucleotide hybridizes to the same target nucleic acid (e.g., a RNA transcript) as another oligonucleotide described herein (e.g., an oligonucleotide in Table 1 (e.g., Table 1A, Table 1B, Table 1C, Table 1D)), wherein the portion of the target nucleic acid to which the oligonucleotide hybridizes to overlaps with the portion to which the another oligonucleotide hybridizes to. In some embodiments, an oligonucleotide hybridizes to the same target nucleic acid (e.g., a RNA transcript) as another oligonucleotide described herein (e.g., an oligonucleotide in Table 1 (e.g., Table 1A, Table 1B, Table 1C, Table 1D)), wherein the portion of the target nucleic acid to which the oligonucleotide hybridizes to is within in a region in the target nucleic acid, wherein the region is about 20-500 (e.g., 25-500, 30-500, 20-400, 20-300, 20-200, 20-200, 20-50, 25-100, 30-100, 50-100, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, etc.) nucleobases in length and comprises the portion to which the another oligonucleotide hybridizes to. In some embodiments, base sequence of the portion in a target nucleic acid to which an oligonucleotide hybridizes to is complementary to base sequence of the oligonucleotide with no mismatches or with one or more (e.g., 1, 2, 3, etc.) mismatches. In some embodiments, the number of mismatches is about or no more than about 1, 2 or 3.

[0124] In some embodiments, a portion in a target nucleic acid to which an oligonucleotide hybridizes to, or to whose sequence the base sequence of an oligonucleotide is complementary to, has the same number of nucleobases as the oligonucleotide. In some embodiments, such a portion has a different number of nucleobases as the oligonucleotide; in some embodiments, the difference is no more than about 1, 2, 3, 4, or 5. In some embodiments, such a portion is of about or at least about 10-100 (e.g., 10-50, 15-100, 15-50, 18-50, 20-50, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.). In some embodiments, such a portion comprises a unique sequence Page 42 of 274 12621671v1Attorney Docket No.: 2010581-1497 element that differentiates the target from a non-target. In some embodiments, such a portion can be in or overlap with any element of a target or a complement thereof, e.g., a promoter region, an enhancer region, a transcriptional stop region, a translational start signal, a translation stop signal, a coding region, a non-coding region, an exon, an intron, an intron / exon junction, a 5' UTR, a 3' UTR, etc. In some embodiments, such a portion is in two or more variants of transcripts of a target nucleic acid, e.g., a gene. In some embodiments, such a portion is only in a single variant. In some embodiments, such a portion can differentiate an allele from another. In some embodiments, such a portion can differentiate an allele from all the others. In some embodiments, such a portion can differentiate a mutation from a wild-type. In some embodiments, such a portion can differentiate one form of a SNP from another or all the other forms of the SNP.

[0125] In some embodiments, a “portion” (e.g., of a base sequence or a pattern of modifications) is about or at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 monomeric units long (e.g., for a base sequence, about or at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases long). In some embodiments, a “portion” of a base sequence or a nucleic acid is about or at least 5 bases long. In some embodiments, it is about or at least about 10 bases long. In some embodiments, it is about or at least about 15 bases long. In some embodiments, it is about or at least about 16 bases long. In some embodiments, it is about or at least about 17 bases long. In some embodiments, it is about or at least about 18 bases long. In some embodiments, it is about or at least about 19 bases long. In some embodiments, it is about or at least about 20 bases long. Lengths

[0126] As appreciated by those skilled in the art, oligonucleotides can be of various lengths to provide desired properties and / or activities for various uses. Many technologies for assessing, selecting and / or optimizing oligonucleotide length are available and can be utilized in accordance with the present disclosure. As demonstrated herein, in many embodiments, oligonucleotides are of suitable lengths to hybridize with their targets and reduce levels of their targets and / or an encoded product thereof. In some embodiments, an oligonucleotide is long enough to recognize a target nucleic acid (e.g., a mRNA). In some embodiments, an oligonucleotide is sufficiently long to distinguish between a target nucleic acid and non-target nucleic acids to reduce off-target effects. In some embodiments, an oligonucleotide is sufficiently short to reduce complexity of manufacture or production and to reduce cost of products.

[0127] In some embodiments, the base sequence of an oligonucleotide is about 10-500 nucleobases in length. In some embodiments, a base sequence is about 10-500 nucleobases in length. In some embodiments, a base sequence is about 10-50 nucleobases in length. In some embodiments, a base sequence is about 15-50 nucleobases in length. In some embodiments, a base sequence is from about 15 to about 30 nucleobases in length. In some embodiments, a base sequence is from about 10 to about 25 nucleobases in length. In some embodiments, a base sequence is from about 15 to about 22 nucleobases in length. In some embodiments, a base sequence is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobases in length. In Page 43 of 274 12621671v1Attorney Docket No.: 2010581-1497 some embodiments, a base sequence is about 18 nucleobases in length. In some embodiments, a base sequence is about 19 nucleobases in length. In some embodiments, a base sequence is about 20 nucleobases in length. In some embodiments, a base sequence is about 21 nucleobases in length. In some embodiments, a base sequence is about 22 nucleobases in length. In some embodiments, a base sequence is about 23 nucleobases in length. In some embodiments, a provided oligonucleotide has a length of no more than about 49, 45, 40, 30, 35, 25, or 23 total nucleotides as described herein. In some embodiments, a base sequence is about 24 nucleobases in length. In some embodiments, a base sequence is about 25 nucleobases in length. In some embodiments, each nucleobase is optionally substituted A, T, C, G, U, or an optionally substituted tautomer of A, T, C, G, or U. Regions, Wings and Cores

[0128] In some embodiments, an oligonucleotide comprises several regions each of which independently comprises one or more consecutive nucleosides and optionally one or more internucleotidic linkages. In some embodiments, a region differs from another in that it is located at a different location in an oligonucleotide. For example, in an oligonucleotide comprising 5’-wing-core-wing-3’, the 5’-wing is located to the 5’ side of the core, the core is in the middle of the two wings, and the 3’-wing is located to the 3’ side of the core. In some embodiments, a region differs from its neighboring region(s) in that it contains one or more structural feature that are different from those corresponding structural features of its neighboring region(s). Example structural features include nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, internucleotidic linkages and patterns thereof (which can be internucleotidic linkage types (e.g., PO linkage, PS linkage, PN linkage, etc.) and patterns thereof, linkage phosphorus modifications (backbone phosphorus modifications) and patterns thereof, backbone chiral center (linkage phosphorus) stereochemistry and patterns thereof (e.g., combination of Rp and / or Sp of chirally controlled internucleotidic linkages (sequentially from 5’ to 3’), optionally with non-chirally controlled internucleotidic linkages and / or natural phosphate linkages, if any (e.g., OSOOO RSSRS SSSRS SOOOS)). In some embodiments, a region comprises a chemical modification (e.g., a sugar modification, base modification, internucleotidic linkage, or stereochemistry of internucleotidic linkage) not present in its neighboring region(s). In some embodiments, a region lacks a chemical modification present in its neighboring regions(s).

[0129] In some embodiments, an oligonucleotide comprises or consists of two or more regions. In some embodiments, an oligonucleotide comprises or consists of three or more regions. In some embodiments, an oligonucleotide comprises or consists of two neighboring regions, wherein one region is designated as a wing region and the other a core region. In some embodiments, an oligonucleotide comprises or consists of a wing- core or core-wing structure. In some embodiments, an oligonucleotide comprises or consists of three neighboring regions, wherein one region is flanked by two neighboring regions. In some embodiments, the middle region is designated as the core region, and each of the flanking region a wing region (a 5’-wing if connected to the 5’-end of the core, a 3’-wing if connected to the 3’-end of the core). In some embodiments, Page 44 of 274 12621671v1Attorney Docket No.: 2010581-1497 an oligonucleotide comprises or consists of a 5’-wing-core-wing-3’ structure. In some embodiments, an oligonucleotide comprises or consists of a 5’-wing-core-3’ structure. In some embodiments, an oligonucleotide comprises or consists of a 5’-core-wing-3’ structure.

[0130] In some embodiments, the present disclosure provides methods for utilizing PN linkages including in core regions.

[0131] In some embodiments, a first region (e.g., a wing) differs from a second region (e.g., a core) in that the first region contains sugar modification(s) or a pattern thereof absent from the second region. In some embodiments, a first (e.g., wing) region comprises a sugar modification(s) or a pattern thereof absent from a second (e.g., core) region. In some embodiments, a sugar modification is a 2’-modification. In some embodiments, a 2’-modification is 2’-ORsa, wherein Rsais optionally substituted C1-6 aliphatic. In some embodiments, a 2’-modification is 2’-ORsa, wherein Rsais optionally substituted C1-6 alkyl. In some embodiments, a 2’-modification is 2’-MOE. In some embodiments, a 2’-modification is 2’-OMe. In some embodiments, a 2’-modification is 2’−O−Ls2c−, wherein the oxygen atom is bonded to the 2’-carbon and Ls2cis bonded to another carbon of the sugar ring, e.g., the 4’-carbon, and Ls2cis an optionally substituted C1-2 aliphatic group. In some embodiments, Ls2cis Ls24, wherein Ls24is optionally substituted −CH2− and is bonded to the 4’- carbon. In some embodiments, Ls24is −CH2− and is bonded to the 4’-carbon. In some embodiments, a modified sugar is a bicyclic sugar, e.g., a LNA sugar. In some embodiments, each sugar in a region is independently modified. In some embodiments, each sugar of a region (e.g., a wing) independently comprises a modification, which can be the same or different from each other. In some embodiments, each sugar of a region (e.g., a wing) comprises the same modification, e.g., 2’-modification as described in the present disclosure. In some embodiments, sugars of a region (e.g., a core) are not modified. In some embodiments, each sugar of a region (e.g., a core) is a non-modified DNA sugar (with two −H at the 2’-position). In some embodiments, the structure of a provided oligonucleotide comprises or consists of a wing-core, core-wing, or 5’-wing-core-wing-3’ structure, wherein each wing independently comprises one or more sugar modifications, and each sugar in the core is a natural DNA sugar (with two −H at the 2’-position).

[0132] Additionally or alternatively, a first region (e.g., a wing) can contain internucleotidic linkage(s) or pattern thereof that differs from another region (e.g., a core or another wing). In some embodiments, a region (e.g., a wing) comprises two or more consecutive natural phosphate linkages. In some embodiments, a region (e.g., a core) comprises no consecutive natural phosphate linkages. In some embodiments, the structure of a provided oligonucleotide comprises or consists of a 5’-wing-core-3’, 5’-core-wing-3’, or 5’-wing-core-wing-3’ structure. In some embodiments, the core comprises no consecutive natural phosphate linkages. In some embodiments, in a 5’-wing-core-wing-3’ structure, each wing independently comprises two or more consecutive internucleotidic linkages. Unless otherwise noted, for the purpose of stereochemistry of 5’-wing- core-wing-3’ structures, internucleotidic linkages connecting a core with a wing are included in the core (e.g., see above).

[0133] In some embodiments, a region is a 5’-wing, a 3’-wing, or a core as described herein. In some Page 45 of 274 12621671v1Attorney Docket No.: 2010581-1497 embodiments, a 5’-wing is at the 5’ end of the oligonucleotide. In some embodiments, a 3’-wing is at the 3’- end of the oligonucleotide. In some embodiments, an oligonucleotide chain comprises a 5’-wing-core-wing-3’ structure. In some embodiments, an oligonucleotide chain consists of a 5’-wing-core-wing-3’ structure. In some embodiments, an oligonucleotide consists of a 5’-wing-core-wing-3’ structure. 5'-Wings

[0134] In some embodiments, a 5’-wing comprises one or more modified sugars. Various modified sugars are described herein and may be utilized in a 5’-wing. In some embodiments, each sugar in a 5’-wing is independently a modified sugar. In some embodiments, a 5’-wing comprises one or more 2’-modified sugars. In some embodiments, each sugar in a 5’-wing is independently a 2’-modified sugar. In some embodiments, a 5’-wing comprises one or more 2’-ORsamodified sugar, wherein Rsais optionally substituted C1-4 aliphatic. In some embodiments, each sugar of a 5’-wing independently comprises a 2’-ORsamodification. In some embodiments, 2’-ORsais 2’-MOE. In some embodiments, each sugar of a 5’-wing comprises 2’-MOE. In some embodiments, a 5’-wing comprises both 2’-OMe and 2’-MOE modified sugars. In some embodiments, the first and the last sugars in a 5’-wing are 2’-OMe modified sugar, and each other sugar in a 5’-wing is a 2’-MOE modified sugar. In some embodiments, a sugar in a 5’-wing comprises −O−Ls2c−, wherein O and Ls2cis independently bonded to a sugar carbon atom, and Ls2cis an optionally substituted bivalent C1-2 aliphatic group. In some embodiments, a 2’-modification is 2’−O−Ls2c−, wherein the oxygen atom is bonded to the 2’-carbon and Ls2cis bonded to another carbon of the sugar ring, e.g., the 4’-carbon, and Ls24is an optionally substituted C1-2 aliphatic group. In some embodiments, Ls2cis Ls24, wherein Ls24is optionally substituted −CH2− and is bonded to the 4’-carbon. In some embodiments, Ls24is −CH2− and is bonded to the 4’-carbon. In some embodiments, Ls24is −CH(CH3)− and is bonded to the 4’-carbon. In some embodiments, Ls24is – (R)−CH(CH3)− and is bonded to the 4’-carbon. In some embodiments, Ls24is −(S)−CH(CH3)− and is bonded to the 4’-carbon.In some embodiments, a modified sugar is a bicyclic sugar, e.g., a LNA sugar, a cEt sugar, a BNA sugar, etc. In some embodiments, a 5’-wing comprises one or more bicyclic sugars.

[0135] Regions, e.g., wings, cores, etc., can be of various suitable lengths. In some embodiments, a region (e.g., a wing, a core, etc.) comprises 1-30, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleobases. In some embodiments, each nucleobase counted in a length, in a region or in an oligonucleotide is independently comprises an optionally substituted monocyclic, bicyclic or polycyclic ring, which ring has at least one nitrogen ring atom. In some embodiments, each nucleobase counted in a length, in a region or in an oligonucleotide is independently optionally substituted A, T, C, G or U, or a substituted tautomer of A, T, C, G or U. In some embodiments, a 5’-wing is about 1-10, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, nucleobases in length. In some embodiments, it is 1 nucleobase in length. In some embodiments, it is about 2 nucleobases in length. In some embodiments, it is about 3 nucleobases in length. In some embodiments, it is about 4 nucleobases in length. In some embodiments, it is about 5 nucleobases in length. In some Page 46 of 274 12621671v1Attorney Docket No.: 2010581-1497 embodiments, it is about 6 nucleobases in length. In some embodiments, it is about 7 nucleobases in length. In some embodiments, each wing of a 5’-wing-core-wing-3’ structure independently has a length as described in the present disclosure. In some embodiments, the two wings are of the same length. In some embodiments, the two wings are of different length. In some embodiments, both wings are 5 nucleobases in length.

[0136] Various internucleotidic linkages can be utilized in a 5’-wing. In some embodiments, a 5’-wing comprises a PO internucleotidic linkage. In some embodiments, a 5’-wing comprises a PS internucleotidic linkage. In some embodiments, a 5’-wing comprises a PN internucleotidic linkage. In some embodiments, a 5’-wing comprises a PO internucleotidic linkage and a PN internucleotidic linkage. In some embodiments, a 5’-wing comprises a PO internucleotidic linkage and a PS internucleotidic linkage. In some embodiments, a 5’-wing comprises a PN internucleotidic linkage and a PS internucleotidic linkage. In some embodiments, a 5’-wing comprises a PO internucleotidic linkage, a PS internucleotidic linkage and a PN internucleotidic linkage. In some embodiments, each internucleotidic linkage in a 5’-wing is independently a PO internucleotidic linkage, a PS internucleotidic linkage, or a PN internucleotidic linkage. In some embodiments, each internucleotidic linkage in a 5’-wing is independently a PO internucleotidic linkage or a PS internucleotidic linkage. In some embodiments, each internucleotidic linkage in a 5’-wing is independently a PO internucleotidic linkage or a PN internucleotidic linkage. In some embodiments, each internucleotidic linkage in a 5’-wing is independently a PN internucleotidic linkage or a PS internucleotidic linkage. In some embodiments, a 5’-wing does not contain a natural phosphate linkage. In some embodiments, a 5’-wing does not contain a natural phosphate linkage. In some embodiments, the number of PO internucleotidic linkages in a 5’-wing is about 1, 2, 3, 4 or 5. In some embodiments, the number of PS internucleotidic linkages in a 5’- wing is about 1, 2, 3, 4 or 5. In some embodiments, the number of PN internucleotidic linkages in a 5’-wing is about 1, 2, 3, 4 or 5. In some embodiments, a 5’-wing comprises two or more consecutive PS internucleotidic linkages. In some embodiments, a 5’-wing comprises two or more consecutive PN internucleotidic linkages. In some embodiments, a 5’-wing comprises a Sp PS internucleotidic linkage. In some embodiments, a 5’-wing comprises a Rp PS internucleotidic linkage. In some embodiments, a 5’-wing comprises a Rp PN internucleotidic linkage. In some embodiments, a 5’-wing comprises a Sp PN internucleotidic linkage. In some embodiments, a PO internucleotidic linkage in a 5’-wing is a natural phosphate linkage. In some embodiments, each PO internucleotidic linkage in a 5’-wing is a natural phosphate linkage. In some embodiments, a PS internucleotidic linkage in a 5'-wing is a phosphorothioate internucleotidic linkage. In some embodiments, each PS in a 5'-wing is independently a phosphorothioate internucleotidic linkage. In some embodiments, a PS internucleotidic linkage in a 5'-wing is a Sp phosphorothioate internucleotidic linkage. In some embodiments, each PS in a 5'-wing is independently a Sp phosphorothioate internucleotidic linkage. In some embodiments, a PN internucleotidic linkage in a 5’-wing is a phosphoryl guanidine internucleotidic linkage. In some embodiments, each PN internucleotidic linkage in a 5’-wing is independently a phosphoryl guanidine internucleotidic linkage. In some embodiments, a PN internucleotidic linkage in a 5’-wing is a n001 internucleotidic linkage. In some embodiments, each PN internucleotidic linkage in a 5’-wing is independently Page 47 of 274 12621671v1Attorney Docket No.: 2010581-1497 a n001 internucleotidic linkage. In some embodiments, a PN internucleotidic linkage is Sp and a PN internucleotidic linkage is Rp. In some embodiments, each PN internucleotidic linkage is Sp. In some embodiments, each PN internucleotidic linkage is Rp. In some embodiments, the pattern of internucleotidic linkages of a 5’-wing comprises SS(O)t, wherein t is 1, 2, 3, 4 or 5, SSrepresents a PS linkage in Sp configuration, and each O independently represents a PO linkage. In some embodiments, the pattern of internucleotidic linkages of a 5’-wing comprises SS(NR)t, wherein t is 1, 2, 3, 4, or 5, SS represents a PS linkage in Sp configuration, and each NR independently represents a PN linkage in Rp configuration. In some embodiments, the pattern of internucleotidic linkages of a 5’-wing comprises SSNR. In some embodiments, the pattern of internucleotidic linkages of a 5’-wing comprises SS(NR)t, wherein t is 1, 2, 3, 4, or 5, SSrepresents a PS linkage in Sp configuration, and each NR independently represents a PN linkage in Rp configuration. In some embodiments, the pattern of internucleotidic linkages of a 5’-wing comprises SSNRO. In some embodiments, the pattern of internucleotidic linkages of a 5’-wing comprises SSNRONR. In some embodiments, a 5’-wing comprises an Sp PS internucleotidic linkage preceding one Rp PN internucleotidic linkage. In some embodiments, a 5’-wing comprises an Sp PS internucleotidic linkage preceding two Rp PN internucleotidic linkages. In some embodiments, a 5’-wing comprises an Sp PS internucleotidic linkage preceding three Rp PN internucleotidic linkages. Cores

[0137] In some embodiments, a core is in a middle of an oligonucleotide, e.g., of a 5’-wing-core-wing-3’ structure. In some embodiments, a core comprises one or more (e.g., 1-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) sugars without 2’-substitution. In some embodiments, a core comprises two or more (e.g., 2-20, 5-15, 5-10, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) consecutive sugars without 2’-substitution. In some embodiments, the number of sugars without 2’- substitution is about 2-20 (e.g., 2-20, 5-15, 5-10, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) or more. In some embodiments, it is about 5-15. In some embodiments, it is about 5. In some embodiments, it is about 6. In some embodiments, it is about 7. In some embodiments, it is about 8. In some embodiments, it is about 9. In some embodiments, it is about 10. In some embodiments, it is about 11. In some embodiments, it is about 12. In some embodiments, it is about 13. In some embodiments, it is about 14. In some embodiments, it is about 15. In some embodiments, it is about 16 or more. In some embodiments, a core comprises one or more (e.g., 1-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) natural DNA sugars (2-deoxy-D-ribose). In some embodiments, a core comprises two or more (e.g., 2-20, 5-15, 5-10, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) consecutive natural DNA sugars. In some embodiments, no sugar in a core comprises 2’-substitution. In some embodiments, each sugar in a core is a natural DNA sugar. In some embodiments, about or at least about 50%, 60%, 66%, 70%, 75%, 80%, 85%, 90% or 95% sugars in a core independently do not have a 2’-substitution. In some embodiments, about or at least about 50%, 60%, 66%, 70%, 75%, 80%, 85%, 90% or 95% sugars in a core are DNA sugars. Page 48 of 274 12621671v1Attorney Docket No.: 2010581-1497 In some embodiments, a core comprises one or more modified sugars as described herein. In some embodiments, the number of modified sugars in a core is about or no more than about 0, 1, 2, 3, 4, or 5. In some embodiments, it is 0. In some embodiments, it is about or no more than about 1. In some embodiments, it is about or no more than about 2. In some embodiments, it is about or no more than about 3. In some embodiments, the number of modified sugars in a core is about or no more than about 5%, 10%, 15%, 20%, 25%, or 30% of sugars in a core. In some embodiments, it is no more than about 10%. In some embodiments, it is no more than about 20%. In some embodiments, it is no more than about 30%. In some embodiments, a modified sugar in a core is a 2’-modified sugar. In some embodiments, it is a 2’-ORsamodified sugar wherein Rsais optionally substituted C1-4 aliphatic. In some embodiments, it is a 2’-MOE modified sugar. In some embodiments, it is a 2’-OMe modified sugar. In some embodiments, it is a −O−Ls2c− modified sugar, wherein O and Ls2cis independently bonded to a sugar carbon atom, and Ls2cis an optionally substituted bivalent C1-2 aliphatic group. In some embodiments, it is a 2’−O−Ls2c− modified sugar, wherein the oxygen atom is bonded to the 2’-carbon and Ls2cis bonded to another carbon of the sugar ring, e.g., the 4’-carbon, and Ls24is an optionally substituted C1-2 aliphatic group. In some embodiments, Ls2cis Ls24, wherein Ls24is optionally substituted −CH2− and is bonded to the 4’-carbon. In some embodiments, Ls24is −CH2− and is bonded to the 4’-carbon. In some embodiments, Ls24is −CH(CH3)− and is bonded to the 4’-carbon. In some embodiments, Ls24is −(R)−CH(CH3)− and is bonded to the 4’-carbon. In some embodiments, Ls24is −(S)−CH(CH3)− and is bonded to the 4’-carbon.In some embodiments, it is a bicyclic sugar, e.g., a LNA sugar, a cEt sugar, a BNA sugar, etc.

[0138] In some embodiments, an oligonucleotide or a core thereof forms a duplex with a target nucleic acid or a portion of and can reduce levels of the target nucleic acid and / or product encoded thereby. In some embodiments, a target nucleic acid is cleaved by RNase H. Ribonuclease H (RNase H, e.g., RNase H1, RNase H2, etc.) has been reported to recognize a structure comprising a hybrid of RNA and DNA (e.g., a heteroduplex), and to cleave the RNA. In some embodiments, an oligonucleotide comprising natural DNA sugars (e.g., in a core region) is capable of hybridizing to a RNA such as a mRNA to form a heteroduplex; and this heteroduplex structure is capable of being recognized by RNase H and the RNA is cleaved by RNase H. In some embodiments, a core of a provided oligonucleotide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive natural DNA sugars, and the core is capable of annealing specifically to a target RNA (e.g., pre-mRNA, mature mRNA, etc.), and the formed structure is capable of being recognized by RNase H and the mRNA is cleaved by RNase H. In some embodiments, a core of a provided oligonucleotide comprises 5 or more consecutive natural DNA sugars. In some embodiments, a core comprise 10 consecutive natural DNA sugars. In some embodiments, an oligonucleotide or a core forms a duplex with a portion of a target nucleic acid, wherein the base sequence of the portion differentiate the target nucleic acid from a non-target.

[0139] In some embodiments, a core is about 5-25, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobases in length. In some embodiments, a core is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more in length. In some embodiments, a core is about 5 nucleobases in length. In some Page 49 of 274 12621671v1Attorney Docket No.: 2010581-1497 embodiments, a core is about 6 nucleobases in length. In some embodiments, a core is about 7 nucleobases in length. In some embodiments, a core is about 8 nucleobases in length. In some embodiments, a core is about 9 nucleobases in length. In some embodiments, a core is about 10 nucleobases in length. In some embodiments, a core is about 11 nucleobases in length. In some embodiments, a core is about 12 nucleobases in length. In some embodiments, a core is about 13 nucleobases in length. In some embodiments, a core is about 14 nucleobases in length. In some embodiments, a core is about 15 nucleobases in length. For example, in some embodiments, 5’-wing-core-wing-3’ from 5’ to 3’ is of 2-9-6, 3-9-3, 3-9-4, 3-9-5, 4-7-4, 4- 9-4, 4-9-5, 4-10-5, 4-11-4, 4-11-5, 5-7-5, 5-8-6, 5-9-3, 5-9-5, 5-10-4, 5-10-5, 6-7-6, 6-8-5, or 6-9-2. In some embodiments, it is 5- 10-5. In some embodiments, each nucleobase counted in a length or in a core is independently comprises an optionally substituted monocyclic, bicyclic, or polycyclic ring, which ring has at least one nitrogen ring atom. In some embodiments, each nucleobase counted in a length or in a core is independently optionally substituted A, T, C, G or U, or a substituted tautomer of A, T, C, G or U.

[0140] Various internucleotidic linkages can be utilized in a core. In some embodiments, a core comprises a PO internucleotidic linkage. In some embodiments, a PO internucleotidic linkage in a core bonds to one or two modified sugars, e.g., 5’-modified sugars as described herein. In some embodiments, a core comprises a PS internucleotidic linkage. In some embodiments, a core comprises a PN internucleotidic linkage. In some embodiments, a core comprises a PO internucleotidic linkage and a PN internucleotidic linkage. In some embodiments, a core comprises a PO internucleotidic linkage and a PS internucleotidic linkage. In some embodiments, a core comprises a PN internucleotidic linkage and a PS internucleotidic linkage. In some embodiments, a core comprises a PO internucleotidic linkage, a PS internucleotidic linkage and a PN internucleotidic linkage. In some embodiments, each internucleotidic linkage in a core is independently a PO internucleotidic linkage, a PS internucleotidic linkage, or a PN internucleotidic linkage. In some embodiments, each internucleotidic linkage in a core is independently a PO internucleotidic linkage or a PS internucleotidic linkage. In some embodiments, each internucleotidic linkage in a core is independently a PO internucleotidic linkage or a PN internucleotidic linkage. In some embodiments, each internucleotidic linkage in a core is independently a PN internucleotidic linkage or a PS internucleotidic linkage. In some embodiments, a core does not contain a natural phosphate linkage. In some embodiments, a core does not contain a natural phosphate linkage. In some embodiments, the number of PO internucleotidic linkages in a core is about 1, 2, 3, 4 or 5. In some embodiments, the number of PS internucleotidic linkages in a core is about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1516, 17, 18, 19, 20. In some embodiments, the number of PN internucleotidic linkages in a core is about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, a core comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) consecutive PS internucleotidic linkages. In some embodiments, a core comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) consecutive PN internucleotidic linkages. In some embodiments, a core comprises a Sp PS internucleotidic linkage. In some embodiments, a core comprises a Rp PS internucleotidic linkage. In some embodiments, a core comprises a Rp PS internucleotidic linkage and a Sp PS internucleotidic linkage. In some Page 50 of 274 12621671v1Attorney Docket No.: 2010581-1497 embodiments, a core comprises more Sp PS internucleotidic linkages than Rp PS internucleotidic linkages. In some embodiments, the number of Rp PS internucleotidic linkages that bond to a core sugar is about or no more than about 0, 1, 2, or 3; in some embodiments, it is about or no more than about 3; in some embodiments, it is about or no more than about 2; in some embodiments, it is about or no more than about 3. In some embodiments, about or no more than about 5%, 10%, 15%, 20%, 25%, 30% or 35% of PS internucleotidic linkage that bond to at least one core sugar is Rp. In some embodiments, about or at least about 65%, 70%, 75%, 80%, 85%, 90% or 95% of PS internucleotidic linkage that bond to at least one core sugar is Sp. In some embodiments, a core comprises a Rp PN internucleotidic linkage. In some embodiments, a core comprises a Sp PN internucleotidic linkage. In some embodiments, a PO internucleotidic linkage in a core is a natural phosphate linkage. In some embodiments, each PO internucleotidic linkage in a core is a natural phosphate linkage. In some embodiments, a PS internucleotidic linkage in a core is a phosphorothioate internucleotidic linkage. In some embodiments, each PS in a core is independently a phosphorothioate internucleotidic linkage. In some embodiments, a PS internucleotidic linkage in a core is a Sp phosphorothioate internucleotidic linkage. In some embodiments, each PS in a core is independently a Sp phosphorothioate internucleotidic linkage. In some embodiments, a PN internucleotidic linkage in a core is a phosphoryl guanidine internucleotidic linkage. In some embodiments, each PN internucleotidic linkage in a core is independently a phosphoryl guanidine internucleotidic linkage. In some embodiments, a PN internucleotidic linkage in a core is a n001 internucleotidic linkage. In some embodiments, each PN internucleotidic linkage in a core is independently a n001 internucleotidic linkage. In some embodiments, a PN internucleotidic linkage is Sp and a PN internucleotidic linkage is Rp. In some embodiments, each PN internucleotidic linkage is Sp. In some embodiments, each PN internucleotidic linkage is Rp.

[0141] Among other things, the present disclosure encompasses that recognition that stereochemical patterns of chiral linkage phosphorus in a core can direct cleavage of a target nucleic acid, e.g., a transcript. In some embodiments, certain patterns can provide higher levels of cleavage, higher cleavage rates, and / or higher selectivity. Various useful stereochemical patterns are described herein for utilization in cores. In some embodiments, a pattern of backbone chiral centers is a pattern as described herein. In some embodiments, it comprise RpSpSp. In some embodiments, a pattern of internucleotidic linkages of a core is a pattern as described herein. In some embodiments, when describing patterns of backbone chiral centers or internucleotidic linkages, each internucleotidic linkage that bonds to a core sugar may be included in pattern for cores. In some embodiments, a pattern of internucleotidic linkages of a core comprises SR(SS)n, wherein n is 2, 3, 4, 5, 6, 7 or 8, SRrepresents a PS linkage in Rp configuration, each SSindependently represents a PS linkage in Sp configuration, and each internucleotidic linkage of the SR(SS)nindependently bonds to a sugar in the core. In some embodiments, it comprises SRSSSS. In some embodiments, it comprises (NR)tSR(SS)n, wherein t is 1, 2, 3, 4, or 5, n is 2, 3, 4, 5, 6, 7 or 8, SRrepresents a PS linkage in Rp configuration, each SSindependently represents a PS linkage in Sp configuration, each NRindependently represents a PN linkage in Rp configuration, and each internucleotidic linkage of the (NR)tSR(SS)nindependently bonds to a sugar in the core. In some embodiments, Page 51 of 274 12621671v1Attorney Docket No.: 2010581-1497 a pattern of internucleotidic linkages of a core comprises (NR)tSRSSSS. In some embodiments, it comprises NRSRSSSS. In some embodiments, it comprises NRNRSRSSSS. In some embodiments, it comprises SRNSSS, wherein NSrepresents a PN linkage in Sp configuration. In some embodiments, it comprises (NR)2SRSSSS. In some embodiments, a pattern of internucleotidic linkages of a core comprises (Ln)m(Ln)tSR(SS)n, wherein m is 1, 2, 3, 4 or 5, t is 1, 2, 3, 4, or 5, n is 2, 3, 4, 5, 6, 7 or 8, each Lnis independently SR, SS, NR or NS, each SR independently represents a PS linkage in Rp configuration, each SSindependently represents a PS linkage in Sp configuration, each NRindependently represents a PN linkage in Rp configuration, each NSindependently represents a PN linkage in Sp configuration, and each internucleotidic linkage of the (Ln)m(Ln)tSR(SS)n independently bonds to a sugar in the core. In some embodiments, it is or comprises (Ln)m(NR)tSR(SS)n. In some embodiments, Lnis SR. In some embodiments, Lnis SS. In some embodiments, Lnis NR. In some embodiments, Lnis NS. In some embodiments, each Lnis independently NR, NS or SS. In some embodiments, each Lnis independently NS or SS. In some embodiments, each Lnis independently NR or NS. In some embodiments, each Lnis independently NS. In some embodiments, each Lnis independently NR. In some embodiments, each Lnis independently SS. In some embodiments, it comprises (NS)m(NR)tSR(SS)n. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, (Ln)m(Ln)tSR(SS)n is or comprises (NS)mNRSRSSSS. In some embodiments, (Ln)m(Ln)tSR(SS)n is or comprises (NS)mNRNRSRSSSS. In some embodiments, (Ln)m(Ln)tSR(SS)n is or comprises NRNRSRSSSS. In some embodiments, (Ln)m(Ln)tSR(SS)n is or comprises NSNRNRSRSSSS. In some embodiments, (Ln)m(Ln)tSR(SS)n is or comprises NSNSNRNRSRSSSS. In some embodiments, (Ln)m(Ln)tSR(SS)n is or comprises NSNSNSNRNRSRSSSS. In some embodiments, (Ln)m(Ln)tSR(SS)n is or comprises NSSRSSSS. In some embodiments, (Ln)m(Ln)tSR(SS)n is or comprises SSNSSRSSSS. In some embodiments, (Ln)m(Ln)tSR(SS)n is or comprises NSSRSSSSSRSSSS. In some embodiments, (Ln)m(Ln)tSR(SS)n is or comprises NRSRSSSS. In some embodiments, (Ln)m(Ln)tSR(SS)n is or comprises SSNRSRSSSS. In some embodiments, the SR between (Ln)m(Ln)t and (SS)n is L1.

[0142] In some embodiments, a core comprises 5’−L-1−N1−L1−N2−L2−N3−L3−3’ wherein each variable is independently as described herein. In some embodiments, a core comprises 5’−N-1−L-1−N1−L1−N2−L2−N3−L3−3’ wherein each variable is independently as described herein. In some embodiments, a core comprises 5’−L-2−N-1−L-1−N1−L1−N2−L2−N3−L3−3’ wherein each variable is independently as described herein. In some embodiments, a core comprises 5’−N-2−L-2−N-1−L-1−N1−L1−N2−L2−N3−L3−3’ wherein each variable is independently as described herein. In some embodiments, a core comprises 5’−L-8−N-7−L-7−N-6−L-6−N-5−L-5−N-4−L-4−N-3−L-3−N-2−L-2−N-1−L-1−N1−L1−N2−L2−N3−L3−3’ wherein each variable is independently as described herein. In some embodiments, a core is 5’−L-8−N-7−L-7−N-6−L-6−N-5−L-5−N-4−L-4−N-3−L-3−N-2−L-2−N-1−L-1−N1−L1−N2−L2−N3−L3−3’ wherein each variable is independently as described herein. 3’-wings

[0143] In some embodiments, a 3’-wing comprises one or more modified sugars. Various modified sugars Page 52 of 274 12621671v1Attorney Docket No.: 2010581-1497 are described herein and may be utilized in a 3’-wing. In some embodiments, each sugar in a 3’-wing is independently a modified sugar. In some embodiments, a 3’-wing comprises one or more 2’-modified sugars. In some embodiments, each sugar in a 3’-wing is independently a 2’-modified sugar. In some embodiments, a 3’-wing comprises one or more 2’-ORsamodified sugar, wherein Rsais optionally substituted C1-4aliphatic. In some embodiments, each sugar of a 3’-wing independently comprises a 2’-ORsamodification. In some embodiments, 2’-ORsais 2’-MOE. In some embodiments, each sugar of a 3’-wing comprises 2’-MOE. In some embodiments, a 3’-wing comprises both 2’-OMe and 2’-MOE modified sugars. In some embodiments, a sugar in a 3’-wing comprises −O−Ls2c−, wherein O and Ls2cis independently bonded to a sugar carbon atom, and Ls2cis an optionally substituted bivalent C1-2 aliphatic group. In some embodiments, a 2’-modification is 2’−O−Ls2c−, wherein the oxygen atom is bonded to the 2’-carbon and Ls2cis bonded to another carbon of the sugar ring, e.g., the 4’-carbon, and Ls2cis an optionally substituted C1-2 aliphatic group. In some embodiments, Ls2cis Ls24, wherein Ls24is optionally substituted −CH2− and is bonded to the 4’-carbon. In some embodiments, Ls24is −CH2− and is bonded to the 4’-carbon. In some embodiments, Ls24is −CH(CH3)− and is bonded to the 4’-carbon. In some embodiments, Ls24is –(R)−CH(CH3)− and is bonded to the 4’-carbon. In some embodiments, Ls24is −(S)−CH(CH3)− and is carbon.In some embodiments, a modified sugaris a bicyclic sugar, e.g., a LNA sugar, a cEt sugar, a sugar, etc. In some embodiments, a 3’-wing comprises one or more bicyclic sugars. In some embodiments, each sugar in a 3’-wing is the same. In some embodiments, each sugar in a 3’-wing is independently a 2’-OMe.

[0144] In some embodiments, the two wings of a 5’-wing-core-wing-3’ structure comprise different patterns of sugar modifications or different sugar modifications (and the oligonucleotide has or comprises an “asymmetric” format). In some embodiments, sugar modifications provide improved stability and / or annealing properties compared to absence of sugar modifications. In some embodiments, an oligonucleotide comprises or consists of an asymmetrical format. In some embodiments, a 5’-wing comprises a 2’-MOE modified sugar, and no sugar in a 3’-wing is a 2’-MOE modified sugar. In some embodiments, a 5’-wing comprises a 2’-MOE modified sugar and each sugar in a 3’-wing is independently a 2’-OMe modified sugar. In some embodiments, each sugar in a 5’-wing is independently a 2’-OMe modified sugar, and a 3’-wing comprises a 2’-MOE modified sugar.

[0145] In some embodiments, a 3’-wing is about 1-10, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, nucleobases in length. In some embodiments, it is 1 nucleobase in length. In some embodiments, it is about 2 nucleobases in length. In some embodiments, it is about 3 nucleobases in length. In some embodiments, it is about 4 nucleobases in length. In some embodiments, it is about 5 nucleobases in length. In some embodiments, it is about 6 nucleobases in length. In some embodiments, it is about 7 nucleobases in length. In some embodiments, the two wings are of the same length. In some embodiments, the two wings are of different length. In some embodiments, both wings are 5 nucleobases in length. In some embodiments, it is 5-10-5. In some embodiments, each nucleobase counted in a length or in a core is independently comprises an optionally substituted monocyclic, bicyclic, or polycyclic ring, which ring has at least one nitrogen ring atom. In some Page 53 of 274 12621671v1Attorney Docket No.: 2010581-1497 embodiments, each nucleobase counted in a length or in a core is independently optionally substituted A, T, C, G or U, or a substituted tautomer of A, T, C, G or U.

[0146] Various internucleotidic linkages can be utilized in a 3’-wing. In some embodiments, a 3’-wing comprises a PO internucleotidic linkage. In some embodiments, a 3’-wing comprises a PS internucleotidic linkage. In some embodiments, a 3’-wing comprises a PN internucleotidic linkage. In some embodiments, a 3’-wing comprises a PO internucleotidic linkage and a PN internucleotidic linkage. In some embodiments, a 3’-wing comprises a PO internucleotidic linkage and a PS internucleotidic linkage. In some embodiments, a 3’-wing comprises a PN internucleotidic linkage and a PS internucleotidic linkage. In some embodiments, a 3’-wing comprises a PO internucleotidic linkage, a PS internucleotidic linkage and a PN internucleotidic linkage. In some embodiments, each internucleotidic linkage in a 3’-wing is independently a PO internucleotidic linkage, a PS internucleotidic linkage, or a PN internucleotidic linkage. In some embodiments, each internucleotidic linkage in a 3’-wing is independently a PO internucleotidic linkage or a PS internucleotidic linkage. In some embodiments, each internucleotidic linkage in a 3’-wing is independently a PO internucleotidic linkage or a PN internucleotidic linkage. In some embodiments, each internucleotidic linkage in a 3’-wing is independently a PN internucleotidic linkage or a PS internucleotidic linkage. In some embodiments, a 3’-wing does not contain a natural phosphate linkage. In some embodiments, a 3’-wing does not contain a natural phosphate linkage. In some embodiments, the number of PO internucleotidic linkages in a 3’-wing is about 1, 2, 3, 4 or 5. In some embodiments, the number of PS internucleotidic linkages in a 3’- wing is about 1, 2, 3, 4 or 5. In some embodiments, the number of PN internucleotidic linkages in a 3’-wing is about 1, 2, 3, 4 or 5. In some embodiments, a 3’-wing comprises two or more consecutive PS internucleotidic linkages. In some embodiments, a 3’-wing comprises two or more consecutive PN internucleotidic linkages. In some embodiments, a 3’-wing comprises a Sp PS internucleotidic linkage. In some embodiments, a 3’-wing comprises a Rp PS internucleotidic linkage. In some embodiments, a 3’-wing comprises a Rp PN internucleotidic linkage. In some embodiments, a 3’-wing comprises a Sp PN internucleotidic linkage. In some embodiments, a PO internucleotidic linkage in a 3’-wing is a natural phosphate linkage. In some embodiments, each PO internucleotidic linkage in a 3’-wing is a natural phosphate linkage. In some embodiments, a PS internucleotidic linkage in a 3’-wing is a phosphorothioate internucleotidic linkage. In some embodiments, each PS in a 3’-wing is independently a phosphorothioate internucleotidic linkage. In some embodiments, a PS internucleotidic linkage in a 3’-wing is a Sp phosphorothioate internucleotidic linkage. In some embodiments, each PS in a 3’-wing is independently a Sp phosphorothioate internucleotidic linkage. In some embodiments, a PN internucleotidic linkage in a 3’-wing is a phosphoryl guanidine internucleotidic linkage. In some embodiments, each PN internucleotidic linkage in a 3’-wing is independently a phosphoryl guanidine internucleotidic linkage. In some embodiments, a PN internucleotidic linkage in a 3’-wing is a n001 internucleotidic linkage. In some embodiments, each PN internucleotidic linkage in a 3’-wing is independently a n001 internucleotidic linkage. In some embodiments, a PN internucleotidic linkage is Sp and a PN internucleotidic linkage is Rp. In some embodiments, each PN internucleotidic linkage is Sp. In some Page 54 of 274 12621671v1Attorney Docket No.: 2010581-1497 embodiments, each PN internucleotidic linkage is Rp. In some embodiments, when the number of PN internucleotidic linkage in a 3’-wing is 1, 2, 3, or 4, there are about or no more than about 1, 2, 3, 4, or 5 PN internucleotidic linkages in a core. In some embodiments, when the number of PN internucleotidic linkage in a 3’-wing is 1, 2, 3, or 4, there are no PN internucleotidic linkages in a core. In some embodiments, the first internucleotidic linkage from the 3’-end is a PN internucleotidic linkage. In some embodiments, it is a Sp PN internucleotidic linkage. In some embodiments, it is a Rp PN internucleotidic linkage. In some embodiments, a pattern of internucleotidic linkages of the 3’-wing comprises (SS)m, wherein m is 1, 2, 3, 4, or 5 and each SSindependently represents a PS linkage in Sp configuration. In some embodiments, a pattern of internucleotidic linkages of a 3’-wing is or comprises (SS)m(NR)t, wherein m is 1, 2, 3, 4 or 5, t is 1, 2, 3, 4, or 5, each SS independently represents a PS linkage in Sp configuration, and each NR independently represents a PN linkage in Rp configuration. In some embodiments, a pattern of internucleotidic linkages of the 3’-wing comprises (SS)mNR, wherein m is 1, 2, 3, or 4, each SS independently represents a PS linkage in Sp configuration, and NR represents a PN linkage in Rp configuration. In some embodiments, a pattern of internucleotidic linkages of the 3’-wing comprises SSNR, wherein SS represents a PS linkage in Sp configuration, NR represents a PN linkage in Rp configuration. In some embodiments, a pattern of internucleotidic linkages of the 3’-wing comprises SSSSNR, wherein each SS independently represents a PS linkage in Sp configuration, and NR represents a PN linkage in Rp configuration. In some embodiments, a pattern of internucleotidic linkages of the 3’-wing comprises SSSSSSNR, wherein each SS independently represents a PS linkage in Sp configuration, and NR represents a PN linkage in Rp configuration. In some embodiments, a pattern of internucleotidic linkages of the 3’-wing comprises SSSSSSSSNR, wherein each SS independently represents a PS linkage in Sp configuration, and NR represents a PN linkage in Rp configuration.

[0147] In some embodiments, there is no PN internucleotidic linkage in a 3’-wing. In some embodiments, each internucleotidic linkage is a 3’-wing is independently a PO internucleotidic linkage or a PS internucleotidic linkage. In some embodiments, each internucleotidic linkage in a 3’-wing is independently a natural phosphate linkage or a phosphorothioate internucleotidic linkage. In some embodiments, each internucleotidic linkage in a 3’-wing is independently a PS internucleotidic linkage. In some embodiments, each internucleotidic linkage in a 3’-wing is independently a phosphorothioate internucleotidic linkage.

[0148] In some embodiments, an oligonucleotide (or a wing, core, region, block or any portion thereof) can be or comprise a modification, a pattern of modifications, an internucleotidic linkage, a pattern of internucleotidic linkages, a pattern of chiral centers, a wing, a core, a block, a region, and / or a format (including an asymmetrical format) described US 9394333, US 9744183, US 9605019, US 9598458, US 9982257, US 10160969, US 10479995, US 2020 / 0056173, US 2018 / 0216107, US 2019 / 0127733, US 10450568, US 2019 / 0077817, US 2019 / 0249173, US 2019 / 0375774, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, WO 2019 / 032612, and / or WO 2020 / 191252, the modifications, patterns of modifications, internucleotidic linkages, patterns of internucleotidic linkages, patterns of chiral centers, wings, cores, blocks, Page 55 of 274 12621671v1Attorney Docket No.: 2010581-1497 regions, and formats of each of which are independently incorporated herein by reference. Internucleotidic Linkages

[0149] In some embodiments, oligonucleotides comprise base modifications, sugar modifications, and / or internucleotidic linkage modifications. Various internucleotidic linkages can be utilized in accordance with the present disclosure to link units comprising nucleobases, e.g., nucleosides. In some embodiments, oligonucleotides comprise one or more modified internucleotidic linkages and one or more natural phosphate linkages. As widely known by those skilled in the art, natural phosphate linkages are widely found in natural DNA and RNA molecules; they have the structure of −OP(O)(OH)O−, connect sugars in the nucleosides in DNA and RNA, and may be in various salt forms, for example, at physiological pH (about 7.4), natural phosphate linkages are predominantly exist in salt forms with the anion being −OP(O)(O−)O−. A modified internucleotidic linkage, or a non-natural phosphate linkage, is an internucleotidic linkage that is not natural phosphate linkage or a salt form thereof. Modified internucleotidic linkages, depending on their structures, may also be in their salt forms. For example, as appreciated by those skilled in the art, phosphorothioate internucleotidic linkages which have the structure of −OP(O)(SH)O− may be in various salt forms, e.g., at physiological pH (about 7.4) with the anion being −OP(O)(S−)O−.

[0150] In some embodiments, a linkage contains a linkage phosphorus atom bonded to an oxygen atom which oxygen atom is not bonded to or is not part of a backbone sugar (“a PO linkage”, e.g., a natural phosphate linkage). In some embodiments, a linkage contains a linkage phosphorus atom bonded to a sulfur atom which sulfur atom is not bonded to or is not part of a backbone sugar (“a PS linkage”, e.g., a phosphorothioate internucleotidic linkage). In some embodiments, a linkage contains a linkage phosphorus atom bonded to a nitrogen atom which nitrogen atom is not bonded to or is not part of a backbone sugar (“a PN linkage”, e.g., n001). In some embodiments, an oligonucleotide comprises one or more PS linkages. In some embodiments, an oligonucleotide comprises one or more PO linkages. In some embodiments, an oligonucleotide comprises one or more PN linkages. In some embodiments, an oligonucleotide comprises one or more PS and one or more PO linkages. In some embodiments, an oligonucleotide comprises one or more PS and one or more PN linkages. In some embodiments, an oligonucleotide comprises one or more PS, one or more PN and one or more PO linkages. In some embodiments, a PO linkage is a natural phosphate linkage. In some embodiments, each PO linkage is independently a natural phosphate linkage. In some embodiments, a PS linkage is a phosphorothioate internucleotidic linkage. In some embodiments, a PS linkage can be converted into a phosphorothioate internucleotidic linkage when administered to a subject. In some embodiments, each PS linkage is independently a phosphorothioate internucleotidic linkage or a linkage that can be converted into a phosphorothioate internucleotidic linkage when administered to a subject. In some embodiments, each PS linkage is independently a phosphorothioate internucleotidic linkage. In some embodiments, a PN linkage is a phosphoramidate linkage. In some embodiments, each PN linkage is independently a phosphoramidate linkage. In some embodiments, a PN linkage is a phosphoryl guanidine linkage. In some embodiments, each PN linkage Page 56 of 274 12621671v1Attorney Docket No.: 2010581-1497 is independently a phosphoryl guanidine linkage. In some embodiments, a PN linkage has the structure of −O−P(O)(−N=C[N(R1)2]2)−O− or a salt form thereof, wherein each R1is independently as described herein. In some embodiments, each PN linkage independently has the structure of −O−P(O)(−N=C[N(R1)2]2)−O− or a salt form thereof, wherein each R1is independently as described herein. In some embodiments, a PN linkage is n001. In some embodiments, each PN linkage is independently n001. In some embodiments, a PN linkage is n006. In some embodiments, each PN linkage is independently n006. In some embodiments, a PN linkage is MsPA. In In some embodiments, each PN linkage is independently MsPA. In some embodiments, a PN linkage is a phosphoramidate linkage. In some embodiments, each PN linkage is independently a phosphoramidate linkage. In some embodiments, a PN linkage is a phosphoryl guanidine linkage. In some embodiments, each PN linkage is independently a phosphoryl guanidine linkage.

[0151] In some embodiments, an oligonucleotide comprises at least two different types of internucleotidic linkages. In some embodiments, an oligonucleotide comprises at least three different types of internucleotidic linkages.

[0152] In some embodiments, an oligonucleotide comprises an internucleotidic linkage which is a modified internucleotidic linkage, e.g., phosphorothioate, phosphorodithioate, methylphosphonate, phosphoroamidate, thiophosphate, 3’-thiophosphate, or 5’-thiophosphate. In some embodiments, a modified internucleotidic linkage is a PS linkage. In some embodiments, a modified linkage is a PN linkage. In some embodiments, a modified internucleotidic linkage is a PO linkage (e.g., other than a natural phosphate linkage). In some embodiments, each modified internucleotidic linkage is independently a PN internucleotidic linkage or a PS internucleotidic linkage.

[0153] In some embodiments, a modified internucleotidic linkage is a chiral internucleotidic linkage which comprises a chiral linkage phosphorus. In some embodiments, a chiral internucleotidic linkage is a phosphorothioate linkage. In some embodiments, a chiral internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, a chiral internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, a chiral internucleotidic linkage is chirally controlled with respect to its chiral linkage phosphorus. In some embodiments, a chiral internucleotidic linkage is stereochemically pure with respect to its chiral linkage phosphorus. In some embodiments, a chiral internucleotidic linkage is not chirally controlled. In some embodiments, a pattern of backbone chiral centers comprises or consists of positions and linkage phosphorus configurations of chirally controlled internucleotidic linkages (Rp or Sp) and positions of achiral internucleotidic linkages (e.g., natural phosphate linkages).

[0154] In some embodiments, an internucleotidic linkage comprises a P-modification, wherein a P- modification is a modification at a linkage phosphorus. In some embodiments, a modified internucleotidic linkage is a moiety which does not comprise a phosphorus but serves to link two sugars or two moieties that each independently comprises a nucleobase, e.g., as in peptide nucleic acid (PNA).

[0155] In some embodiments, an oligonucleotide comprises a modified internucleotidic linkage, e.g., those having the structure of Formula I, I-a, I-b, or I-c and described herein and / or in: WO 2018 / 022473, WO Page 57 of 274 12621671v1Attorney Docket No.: 2010581-1497 2018 / 098264, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, WO 2019 / 032612, WO 2020 / 191252, WO 2021 / 071858, and / or WO 2022 / 099159, the internucleotidic linkages (e.g., those of Formula I, I-a, I-b, I-c, etc.) of each of which are independently incorporated herein by reference. In some embodiments, a modified internucleotidic linkage is a chiral internucleotidic linkage. In some embodiments, a modified internucleotidic linkage is a phosphorothioate internucleotidic linkage.

[0156] In some embodiments, a modified internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, provided oligonucleotides comprise one or more non- negatively charged internucleotidic linkages. In some embodiments, a non-negatively charged internucleotidic linkage is a positively charged internucleotidic linkage. In some embodiments, a non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, the present disclosure provides oligonucleotides comprising one or more neutral internucleotidic linkages. In some embodiments, a non-negatively charged internucleotidic linkage has the structure of Formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a- 1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., or a salt form thereof, as described herein and / or in US 9394333, US 9744183, US 9605019, US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017 / 062862, WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679, WO 2017 / 210647, WO 2018 / 098264, WO 2018 / 022473, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 032612, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, WO 2019 / 032612, WO 2020 / 191252, WO 2021 / 071858, and / or WO 2022 / 099159, the non- negatively charged internucleotidic linkages (e.g., those of Formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., or a suitable salt form thereof) of each of which are independently incorporated herein by reference.

[0157] In some embodiments, a non-negatively charged internucleotidic linkage can improve the delivery and / or activities.

[0158] In some embodiments, a modified internucleotidic linkage (e.g., a non-negatively charged internucleotidic linkage) comprises optionally substituted triazolyl. In some embodiments, a modified internucleotidic linkage (e.g., a non-negatively charged internucleotidic linkage) comprises optionally substituted alkynyl. In some embodiments, a modified internucleotidic linkage comprises a triazole or alkyne moiety. In some embodiments, a triazole moiety, e.g., a triazolyl group, is optionally substituted. In some embodiments, a triazole moiety, e.g., a triazolyl group) is substituted. In some embodiments, a triazole moiety is unsubstituted. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted cyclic guanidine moiety. In some embodiments, a modified internucleotidic linkage has the structure of optionally chirally controlled, wherein R1is −L−R’, wherein L is LBas describedPage 58 of 274 12621671v1Attorney Docket No.: 2010581-1497 herein, and R’ is as described herein. In some embodiments, each R1is independently R’. In some embodiments, each R’ is independently R. In some embodiments, two R1are R and are taken together to form a ring as described herein. In some embodiments, two R1on two different nitrogen atoms are R and are taken together to form a ring as described herein. In some embodiments, R1is independently optionally substituted C1-6 aliphatic as described herein. In some embodiments, R1is methyl. In some embodiments, two R’ on the same nitrogen atom are R and are taken together to form a ring as described herein. In some embodiments, a modified internucleotidic linkage has the is optionally chirally controlled. Insome In some embodiments, a modified internucleotidiclinkage comprises an optionally substituted cyclic guanidine moiety and has the structureS. In some embodiments, W is O. In somecharged internucleotidic linkage is stereochemically controlled.

[0159] In some embodiments, a non-negatively charged internucleotidic linkage or a neutral internucleotidic linkage is an internucleotidic linkage comprising a triazole moiety. In some embodiments, a non-negatively charged internucleotidic linkage or a non-negatively charged internucleotidic linkage comprises an optionally substituted triazolyl group. In some embodiments, an internucleotidic linkage comprising a triazole moiety (e.g., an optionally substituted triazolyl group) has the . In someembodiments, an internucleotidic linkage comprising a triazole moiety has the structure . In some embodiments, an internucleotidic linkage comprising a triazoleof Page 59 of 274 12621671v1Attorney Docket No.: 2010581-1497 some embodiments, an internucleotidic linkage comprising analkyne moiety (e.g., an optionally substituted alkynyl group) has the , wherein W is O or S. In some embodiments, an internucleotidic linkage, e.g., a non- internucleotidiclinkage, a neutral internucleotidic linkage, comprises a cyclic guanidine embodiments, an internucleotidic linkage comprising a cyclic guanidine moiety has the embodiments, a non-negatively charged internucleotidic linkage, or a orcomprising a structure selected from , or, wherein W is O or S.

[0160] In some embodiments, an internucleotidic linkage comprises a Tmg ). In someembodiments, an internucleotidic linkage comprises a Tmg group and has(the “Tmg internucleotidic linkage”). In some embodiments, neutralinternucleotidic linkages of PNA and PMO, and an Tmg internucleotidic linkage.

[0161] In some embodiments, a non-negatively charged internucleotidic linkage has the structure of Formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., or a salt form thereof. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is Page 60 of 274 12621671v1Attorney Docket No.: 2010581-1497 nitrogen. In some embodiments, such a heterocyclyl or heteroaryl group is of a 5-membered ring. In some embodiments, such a heterocyclyl or heteroaryl group is of a 6-membered ring.

[0162] In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-6 membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a heteroaryl group is directly bonded to a linkage phosphorus. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted triazolyl group. In some embodiments, a non-negatively charged internucleotidic linkage comprises an unsubstituted triazolyl group, e.g., . In some embodiments, a non-negatively charged internucleotidic linkage comprises a substituted triazolyl group, .

[0163] In some embodiments, a non-negatively charged internucleotidic linkagesubstituted 5-20 membered heterocyclyl group having 1-10 heteroatoms. In some embodiments, a non- negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heterocyclyl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a non- negatively charged internucleotidic linkage comprises an optionally substituted 5-6 membered heterocyclyl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a non- negatively charged internucleotidic linkage comprises an optionally substituted 5-membered heterocyclyl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, at least two heteroatoms are nitrogen. In some embodiments, a heterocyclyl group is directly bonded to a linkage phosphorus. In some embodiments, a heterocyclyl group is bonded to a linkage phosphorus through a linker, e.g., =N− when the heterocyclyl group is part of a guanidine moiety who directed bonded to a linkage phosphorus through its =N−. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted group. In some embodiments, a non-negatively charged internucleotidic linkage comprises an substituted group. In some embodiments, a non-negatively Page 61 of 274 12621671v1Attorney Docket No.: 2010581-1497 charged internucleotidic linkage group, wherein each R1is independently −L−R. In some embodiments, each R1is substituted C1-6 alkyl. In some embodiments, each R1is independently methyl.

[0164] In some embodiments, a modified internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage, comprises a triazole or alkyne moiety, each of which is optionally substituted. In some embodiments, a modified internucleotidic linkage comprises a triazole moiety. In some embodiments, a modified internucleotidic linkage comprises a unsubstituted triazole moiety. In some embodiments, a modified internucleotidic linkage comprises a substituted triazole moiety. In some embodiments, a modified internucleotidic linkage comprises an alkyl moiety. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted alkynyl group. In some embodiments, a modified internucleotidic linkage comprises an unsubstituted alkynyl group. In some embodiments, a modified internucleotidic linkage comprises a substituted alkynyl group. In some embodiments, an alkynyl group is directly bonded to a linkage phosphorus.

[0165] In some embodiments, an oligonucleotide comprises different types of internucleotidic phosphorus linkages. In some embodiments, a chirally controlled oligonucleotide comprises at least one natural phosphate linkage and at least one modified (non-natural) internucleotidic linkage. In some embodiments, an oligonucleotide comprises at least one natural phosphate linkage and at least one phosphorothioate. In some embodiments, an oligonucleotide comprises at least one non-negatively charged internucleotidic linkage. In some embodiments, an oligonucleotide comprises at least one natural phosphate linkage and at least one non- negatively charged internucleotidic linkage. In some embodiments, an oligonucleotide comprises at least one phosphorothioate internucleotidic linkage and at least one non-negatively charged internucleotidic linkage. In some embodiments, an oligonucleotide comprises at least one phosphorothioate internucleotidic linkage, at least one natural phosphate linkage, and at least one non-negatively charged internucleotidic linkage. In some embodiments, oligonucleotides comprise one or more, e.g., 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more non-negatively charged internucleotidic linkages. In some embodiments, oligonucleotides comprise no more than a certain number of non-negatively charged internucleotidic linkages, e.g., no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 non- negatively charged internucleotidic linkages. In some embodiments, oligonucleotides comprise no non- negatively charged internucleotidic linkages. In some embodiments, a non-negatively charged internucleotidic Page 62 of 274 12621671v1Attorney Docket No.: 2010581-1497 linkage is not negatively charged in that at a given pH in an aqueous solution less than 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the internucleotidic linkage exists in a negatively charged salt form. In some embodiments, a pH is about pH 7.4. In some embodiments, a pH is about 4-9. In some embodiments, the percentage is less than 10%. In some embodiments, the percentage is less than 5%. In some embodiments, the percentage is less than 1%. In some embodiments, an internucleotidic linkage is a non-negatively charged internucleotidic linkage in that the neutral form of the internucleotidic linkage has no pKa that is no more than about 1, 2, 3, 4, 5, 6, or 7 in water. In some embodiments, no pKa is 7 or less. In some embodiments, no pKa is 6 or less. In some embodiments, no pKa is 5 or less. In some embodiments, no pKa is 4 or less. In some embodiments, no pKa is 3 or less. In some embodiments, no pKa is 2 or less. In some embodiments, no pKa is 1 or less. In some embodiments, pKa of the neutral form of an internucleotidic linkage can be represented by pKa of the neutral form of a compound having the structure of CH3−the internucleotidic linkage−CH3. For example, pKa of the neutral form of an internucleotidic linkage having the structure of Formula I may be represented by the pKa of the neutral form of a compound having the structure of (wherein each of X, Y, Z is can be represented bysome embodiments, a non-negatively charged internucleotidic linkage is a neutralsome embodiments, a non-negatively charged internucleotidic linkage is a positively-charged internucleotidic linkage. In some embodiments, a non-negatively charged internucleotidic linkage comprises a guanidine moiety. In some embodiments, a non-negatively charged internucleotidic linkage comprises a heteroaryl base moiety. In some embodiments, a non-negatively charged internucleotidic linkage comprises a triazole moiety. In some embodiments, a non-negatively charged internucleotidic linkage comprises an alkynyl moiety.

[0166] In some embodiments, a neutral or non-negatively charged internucleotidic linkage has the structure of any neutral or non-negatively charged internucleotidic linkage described in any of: US 9394333, US 9744183, US 9605019, US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017 / 062862, WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679, WO 2017 / 210647, WO 2018 / 098264, WO 2018 / 022473, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 032612, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, WO 2019 / 032612, WO 2020 / 191252, WO 2021 / 071858, and / or WO 2022 / 099159, each neutral or non-negatively charged internucleotidic linkage of each of which is hereby incorporated by reference.

[0167] In some embodiments, each R’ is independently optionally substituted C1-6 aliphatic. In some Page 63 of 274 12621671v1Attorney Docket No.: 2010581-1497 embodiments, each R’ is independently optionally substituted C1-6alkyl. In some embodiments, each R’ is independently −CH3. In some embodiments, each Rsis −H.

[0168] In some embodiments, a non-negatively charged internucleotidic linkage has the structure of some embodiments, a non-negatively charged internucleotidic linkage has the structure . In some embodiments, a non-negatively charged internucleotidic linkage has thestructure some embodiments, a non-negatively charged internucleotidic linkage hasthe some embodiments, a non-negatively charged internucleotidic linkagehas the structure . In some embodiments, a non-negatively charged internucleotidiclinkage has the structure . In some embodiments, a non-negatively chargedinternucleotidic linkage has the . In some embodiments, a non-negativelycharged internucleotidic linkage has the . In some embodiments, a non-negatively charged internucleotidic linkage has the In some embodiments,Page 64 of 274 12621671v1Attorney Docket No.: 2010581-1497 O a non-negatively charged internucleotidic linkage has the In some embodiments, anon-negatively charged internucleotidic linkage has the In some embodiments, anon-negatively charged internucleotidic linkage has the In some embodiments, W is O. In some embodiments, W is S. In some linkage is a non-negatively charged internucleotidic linkage described above.

[0169] In some embodiments, provided oligonucleotides comprise 1 or more internucleotidic linkages of Formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2, which are described in US 9394333, US 9744183, US 9605019, US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017 / 062862, WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679, WO 2017 / 210647, WO 2018 / 098264, WO 2018 / 022473, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 032612, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, WO 2019 / 032607, WO 2019 / 032612, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, WO 2019 / 032612, WO 2020 / 191252, WO 2021 / 071858, and / or WO 2022 / 099159, the Formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2, or salt forms thereof of each of which are independently incorporated herein by reference.

[0170] In some embodiments, an oligonucleotide comprises a neutral internucleotidic linkage and a chirally controlled internucleotidic linkage. In some embodiments, an oligonucleotide comprises a neutral internucleotidic linkage and a chirally controlled internucleotidic linkage which is not the neutral internucleotidic linkage. In some embodiments, an oligonucleotide comprises a neutral internucleotidic linkage and a chirally controlled phosphorothioate internucleotidic linkage. In some embodiments, the present disclosure provides an oligonucleotide comprising one or more non-negatively charged internucleotidic linkages and one or more phosphorothioate internucleotidic linkages, wherein each phosphorothioate internucleotidic linkage in the oligonucleotide is independently a chirally controlled internucleotidic linkage. In some embodiments, the present disclosure provides an oligonucleotide comprising one or more neutral internucleotidic linkages and one or more phosphorothioate internucleotidic linkage, wherein each phosphorothioate internucleotidic linkage in the oligonucleotide is independently a chirally controlled internucleotidic linkage. In some embodiments, an oligonucleotide comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more chirally controlled phosphorothioate internucleotidic linkages. In some Page 65 of 274 12621671v1Attorney Docket No.: 2010581-1497 embodiments, non-negatively charged internucleotidic linkage is chirally controlled. In some embodiments, non-negatively charged internucleotidic linkage is not chirally controlled. In some embodiments, a neutral internucleotidic linkage is chirally controlled. In some embodiments, a neutral internucleotidic linkage is not chirally controlled. In some embodiments, an oligonucleotide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) chirally controlled and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) non-chirally controlled chiral internucleotidic linkages. In some embodiments, an oligonucleotide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) chirally controlled and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) non-chirally controlled non-negatively charged internucleotidic linkages (in some embodiments, each of which is independently n001). In some embodiments, a neutral internucleotidic linkage is chirally controlled. In some embodiments, a neutral internucleotidic linkage is not chirally controlled. In some embodiments, an oligonucleotide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) chirally controlled and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) non-chirally controlled neutral internucleotidic linkages (in some embodiments, each of which is independently n001).

[0171] Without wishing to be bound by any particular theory, the present disclosure notes that a neutral internucleotidic linkage can be more hydrophobic than a phosphorothioate internucleotidic linkage, which can be more hydrophobic than a natural phosphate linkage. Typically, unlike a phosphorothioate internucleotidic linkage or a natural phosphate linkage, a neutral internucleotidic linkage bears less charge. Without wishing to be bound by any particular theory, the present disclosure notes that incorporation of one or more neutral internucleotidic linkages into an oligonucleotide may increase oligonucleotides’ ability to be taken up by a cell and / or to escape from endosomes. Without wishing to be bound by any particular theory, the present disclosure notes that incorporation of one or more neutral internucleotidic linkages can be utilized to modulate melting temperature of duplexes formed between an oligonucleotide and its target nucleic acid.

[0172] Without wishing to be bound by any particular theory, the present disclosure notes that incorporation of one or more non-negatively charged internucleotidic linkages, e.g., neutral internucleotidic linkages, into an oligonucleotide may be able to increase the oligonucleotide’s ability to mediate a function such as reduction of levels of a target nucleic acid and / or a product encoded thereby.

[0173] As appreciated by those skilled in the art, internucleotidic linkages such as natural phosphate linkages and those of Formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II- c-2, II-d-1, II-d-2, or salt forms thereof typically connect two nucleosides (which can either be natural or modified) as described in US 9394333, US 9744183, US 9605019, US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO 2017 / 062862, WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679, WO 2017 / 210647, WO 2018 / 098264, WO 2018 / 022473, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 032612, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, WO 2019 / 032612, WO 2020 / 191252, WO 2021 / 071858, and / or WO 2022 / 099159, the Formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or salt forms thereof of each of which are independently incorporated herein by Page 66 of 274 12621671v1Attorney Docket No.: 2010581-1497 reference. A typical connection, as in natural DNA and RNA, is that an internucleotidic linkage forms bonds with two sugars (which can be either unmodified or modified as described herein). In many embodiments, as exemplified herein an internucleotidic linkage forms bonds through its oxygen atoms or heteroatoms (e.g., Y and Z in various formulae) with one optionally modified ribose or deoxyribose at its 5’ carbon, and the other optionally modified ribose or deoxyribose at its 3’ carbon. In some embodiments, each nucleoside units connected by an internucleotidic linkage independently comprises a nucleobase which is independently an optionally substituted A, T, C, G, or U, or a substituted tautomer of A, T, C, G or U, or a nucleobase comprising an optionally substituted heterocyclyl and / or a heteroaryl ring having at least one nitrogen atom.

[0174] In some embodiments, a linkage has the structure of or comprises −Y−PL(−X−RL)−Z−, or a salt form thereof, wherein: PLis P, P(=W), P−>B(–LL–RL)3, or PN; W is O, N(–LL–RL), S or Se; PNis P=N−C(–LL–R’)(=LN−R’) or P=N–LL–RL; LNis =N−LL1−, =CH−LL1− wherein CH is optionally substituted, or =N+(R’)(Q−)−LL1−; Q−is an anion; each of X, Y and Z is independently –O–, –S–, −LL−N(–LL–RL)−LL−, −LL−N=C(–LL–RL)−LL−, or LL; each RLis independently −LL–N(R’)2, –LL–R’, −N=C(–LL–R’)2, −LL–N(R’)C(NR’)N(R’)2, −LL– N(R’)C(O)N(R’)2, a carbohydrate, or one or more additional chemical moieties optionally connected through a linker; each of LL1and LLis independently L; −CyIL− is −Cy−; each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C1-30 aliphatic group and a C1-30 heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C1-6 alkylene, C1-6 alkenylene, , a bivalent C1–C6 heteroaliphatic group having 1-5 heteroatoms, −C(R’)2−, −Cy−, −O−, −S−, −S−S−, −N(R’)−, −C(O)−, −C(S)−, −C(NR’)−, −C(NR’)N(R’)−, −N(R’)C(NR’)N(R’)−, −C(O)N(R’)−, −N(R’)C(O)N(R’)−, −N(R’)C(O)O−, −S(O)−, −S(O)2−, −S(O)2N(R’)−, −C(O)S−, −C(O)O−, −P(O)(OR’)−, −P(O)(SR’)−, −P(O)(R’)−, −P(O)(NR’)−, −P(S)(OR’)−, −P(S)(SR’)−, −P(S)(R’)−, −P(S)(NR’)−, −P(R’)−, −P(OR’)−, −P(SR’)−, −P(NR’)−, −P(OR’)[B(R’)3]−, −OP(O)(OR’)O−, −OP(O)(SR’)O−, −OP(O)(R’)O−, −OP(O)(NR’)O−, −OP(OR’)O−, −OP(SR’)O−, −OP(NR’)O−, −OP(R’)O−, −OP(OR’)[B(R’)3]O−, and −[C(R’)2C(R’)2O]n−, wherein n is 1- 50, and one or more nitrogen or carbon atoms are optionally and independently replaced with CyL; each −Cy− is independently an optionally substituted bivalent 3-30 membered, monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms; each CyLis independently an optionally substituted trivalent or tetravalent, 3-30 membered, Page 67 of 274 12621671v1Attorney Docket No.: 2010581-1497 monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms; each R’ is independently −R, −C(O)R, −C(O)N(R)2, −C(O)OR, or −S(O)2R; each R is independently −H, or an optionally substituted group selected from C1-30aliphatic, C1-30heteroaliphatic having 1-10 heteroatoms, C6-30aryl, C6-30arylaliphatic, C6-30arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1- 10 heteroatoms, or two R groups are optionally and independently taken together to form a covalent bond, or: two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms; or two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms.

[0175] In some embodiments, an internucleotidic linkage has the structure of −O−PL(−X−RL)−O−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −O−P(=W)(−X−RL)−O−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −O−P(=W)[−N(–LL–RL)−RL]−O−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −O−P(=W)(−NH–LL–RL)−O−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −O−P(=W)[−N(R’)2]−O−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −O−P(=W)(−NHR’)−O−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −O−P(=W)(−NHSO2R)−O−, wherein each variable is independently as described herein. In some embodiments, R is methyl. In some embodiments, an internucleotidic linkage is −O−P(=O)(−NHSO2CH3)−O−. In some embodiments, an internucleotidic linkage has the structure of −O−P(=W)[−N=C(–LL–R’)2]−O−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −O−P(=W)[−N=C[N(R’)2]2]−O−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −OP(=W)(−N=C(R”)2)−O−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −OP(=W)(−N(R”)2)−O−, wherein each variable is independently as described herein. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, such an internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, such an internucleotidic linkage is a neutral internucleotidic linkage.

[0176] In some embodiments, an internucleotidic linkage has the structure of −PL(−X−RL)−Z−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the Page 68 of 274 12621671v1Attorney Docket No.: 2010581-1497 structure of −PL(−X−RL)−O−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=W)(−X−RL)−O−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=W)[−N(–LL–RL)−RL]−O−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=W)(−NH–LL–RL)−O−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=W)[−N(R’)2]−O−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=W)(−NHR’)−O−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=W)(−NHSO2R)−O−, wherein each variable is independently as described herein. In some embodiments, R is methyl. In some embodiments, an internucleotidic linkage is −P(=O)(−NHSO2CH3)−O−. In some embodiments, an internucleotidic linkage has the structure of −P(=W)[−N=C(–LL–R’)2]−O−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=W)[−N=C[N(R’)2]2]−O−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=W)(−N=C(R”)2)−O−, wherein each variable is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=W)(−N(R”)2)−O−, wherein each variable is independently as described herein. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, such an internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, such an internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, P of such an internucleotidic linkage is bonded to N of a sugar.

[0177] In some embodiments, a linkage is a phosphoryl guanidine internucleotidic linkage. In some embodiments, a linkage is a thio-phosphoryl guanidine internucleotidic linkage.

[0178] In some embodiments, one or more methylene units are optionally and independently replaced with a moiety as described herein. In some embodiments, L or LLis or comprises −SO2−. In some embodiments, L or LLis or comprises −SO2N(R’)−. In some embodiments, L or LLis or comprises −C(O)−. In some embodiments, L or LLis or comprises −C(O)O−. In some embodiments, L or LLis or comprises −C(O)N(R’)−. In some embodiments, L or LLis or comprises −P(=W)(R’)−. In some embodiments, L or LLis or comprises −P(=O)(R’)−. In some embodiments, L or LLis or comprises −P(=S)(R’)−. In some embodiments, L or LLis or comprises −P(R’)−. In some embodiments, L or LLis or comprises −P(=W)(OR’)−. In some embodiments, L or LLis or comprises −P(=O)(OR’)−. In some embodiments, L or LLis or comprises −P(=S)(OR’)−. In some embodiments, L or LLis or comprises −P(OR’)−.

[0179] In some embodiments, −X−RLis −N(R’)SO2RL. In some embodiments, −X−RLis −N(R’)C(O)RL. In some embodiments, −X−RLis −N(R’)P(=O)(R’)RL.

[0180] In some embodiments, a linkage, e.g., a non-negatively charged internucleotidic linkage or neutral internucleotidic linkage, has the structure of or comprises −P(=W)(−N=C(R”)2)−, −P(=W)(−N(R’)SO2R”)−, −P(=W)(−N(R’)C(O)R”)−, −P(=W)(−N(R”)2)−, −P(=W)(−N(R’)P(O)(R”)2)−, −OP(=W)(−N=C(R”)2)O−, Page 69 of 274 12621671v1Attorney Docket No.: 2010581-1497 −OP(=W)(−N(R’)SO2R”)O−, −OP(=W)(−N(R’)C(O)R”)O−, −OP(=W)(−N(R”)2)O−, −OP(=W)(−N(R’)P(O)(R”)2)O−, −P(=W)(−N=C(R”)2)O−, −P(=W)(−N(R’)SO2R”)O−, −P(=W)(−N(R’)C(O)R”)O−, −P(=W)(−N(R”)2)O−, or −P(=W)(−N(R’)P(O)(R”)2)O−, or a salt form thereof, wherein: W is O or S; each R” is independently R’, −OR’, −P(=W)(R’)2, or −N(R’)2; each R’ is independently −R, −C(O)R, −C(O)N(R)2, −C(O)OR, or −S(O)2R; each R is independently −H, or an optionally substituted group selected from C1-30 aliphatic, C1-30 heteroaliphatic having 1-10 heteroatoms, C6-30 aryl, C6-30 arylaliphatic, C6-30 arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1- 10 heteroatoms, or two R groups are optionally and independently taken together to form a covalent bond, or: two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms; or two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms.

[0181] In some embodiments, W is O. In some embodiments, an internucleotidic linkage has the structure of −P(=O)(−N=C(R”)2)−, −P(=O)(−N(R’)SO2R”)−, −P(=O)(−N(R’)C(O)R”)−, −P(=O)(−N(R”)2)−, −P(=O)(−N(R’)P(O)(R”)2)−, −OP(=O)(−N=C(R”)2)O−, −OP(=O)(−N(R’)SO2R”)O−, −OP(=O)(−N(R’)C(O)R”)O−, −OP(=O)(−N(R”)2)O−, −OP(=O)(−N(R’)P(O)(R”)2)O−, −P(=O)(−N=C(R”)2)O−, −P(=O)(−N(R’)SO2R”)O−, −P(=O)(−N(R’)C(O)R”)O−, −P(=O)(−N(R”)2)O−, or −P(=O)(−N(R’)P(O)(R”)2)O−, or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of −P(=O)(−N=C(R”)2)− −P(=O)(−N(R”)2)−, −OP(=O)(−N=C(R”)2)−O−, −OP(=O)(−N(R”)2)−O−, −P(=O)(−N=C(R”)2)−O− or −P(=O)(−N(R”)2)−O− or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of −OP(=O)(−N=C(R”)2)−O− or −OP(=O)(−N(R”)2)−O−, or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of −OP(=O)(−N=C(R”)2)−O−, or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of −OP(=O)(−N(R”)2)−O−, or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of −OP(=O)(−N(R’)SO2R”)O−, or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of −OP(=O)(−N(R’)C(O)R”)O−, or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of −OP(=O)(−N(R’)P(O)(R”)2)O−, or a salt form thereof. In some embodiments, a internucleotidic linkage is n001.

[0182] In some embodiments, W is S. In some embodiments, an internucleotidic linkage has the structure of −P(=S)(−N=C(R”)2)−, −P(=S)(−N(R’)SO2R”)−, −P(=S)(−N(R’)C(O)R”)−, −P(=S)(−N(R”)2)−, Page 70 of 274 12621671v1Attorney Docket No.: 2010581-1497 −P(=S)(−N(R’)P(O)(R”)2)−, −OP(=S)(−N=C(R”)2)O−, −OP(=S)(−N(R’)SO2R”)O−, −OP(=S)(−N(R’)C(O)R”)O−, −OP(=S)(−N(R”)2)O−, −OP(=S)(−N(R’)P(O)(R”)2)O−, −P(=S)(−N=C(R”)2)O−, −P(=S)(−N(R’)SO2R”)O−, −P(=S)(−N(R’)C(O)R”)O−, −P(=S)(−N(R”)2)O−, or −P(=S)(−N(R’)P(O)(R”)2)O−, or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of −P(=S)(−N=C(R”)2)− −P(=S)(−N(R”)2)−, −OP(=S)(−N=C(R”)2)−O−, −OP(=S)(−N(R”)2)−O−, −P(=S)(−N=C(R”)2)−O− or −P(=S)(−N(R”)2)−O− or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of −OP(=S)(−N=C(R”)2)−O− or −OP(=S)(−N(R”)2)−O−, or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of −OP(=S)(−N=C(R”)2)−O−, or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of −OP(=S)(−N(R”)2)−O−, or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of −OP(=S)(−N(R’)SO2R”)O−, or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of −OP(=S)(−N(R’)C(O)R”)O−, or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of −OP(=S)(−N(R’)P(O)(R”)2)O−, or a salt form thereof. In some embodiments, an internucleotidic linkage is *n001.

[0183] In some embodiments, an internucleotidic linkage has the structure of −P(=O)(−N(R’)SO2R”)−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=S)(−N(R’)SO2R”)−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=O)(−N(R’)SO2R”)O−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=S)(−N(R’)SO2R”)O−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −OP(=O)(−N(R’)SO2R”)O−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −OP(=S)(−N(R’)SO2R”)O−, wherein R” is as described herein. In some embodiments, R’, e.g., of −N(R’)−, is hydrogen or optionally substituted C1-6 aliphatic. In some embodiments, R’ is C1-6 alkyl. In some embodiments, R’ is hydrogen. In some embodiments, R”, e.g., in −SO2R”, is R’ as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=O)(−NHSO2R”)−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=S)(−NHSO2R”)−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=O)(−NHSO2R”)O−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=S)(−NHSO2R”)O−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −OP(=O)(−NHSO2R”)O−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −OP(=S)(−NHSO2R”)O−, wherein R” is as described herein. In some embodiments, −X−RLis −N(R’)SO2RL, wherein each of R’ and RLis independently as described herein. In some embodiments, RLis R”. In some embodiments, RLis R’. In some embodiments, −X−RLis −N(R’)SO2R”, wherein R’ is as described herein. In some embodiments, −X−RLis −N(R’)SO2R’, wherein R’ is as described herein. In some embodiments, −X−RLis −NHSO2R’, wherein R’ is as described herein. In some embodiments, R’ is R as described herein. In some embodiments, R’ is optionally substituted Page 71 of 274 12621671v1Attorney Docket No.: 2010581-1497 C1-6aliphatic. In some embodiments, R’ is optionally substituted C1-6alkyl. In some embodiments, R’ is optionally substituted phenyl. In some embodiments, R’ is optionally substituted heteroaryl. In some embodiments, R”, e.g., in −SO2R”, is R. In some embodiments, R is an optionally substituted group selected from C1-6aliphatic, aryl, heterocyclyl, and heteroaryl. In some embodiments, R is optionally substituted C1-6aliphatic. In some embodiments, R is optionally substituted C1-6 alkyl. In some embodiments, R is optionally substituted C1-6alkenyl. In some embodiments, R is optionally substituted C1-6alkynyl. In some embodiments, R is optionally substituted methyl. In some embodiments, −X−RLis −NHSO2CH3. In some embodiments, R is −CF3. In some embodiments, R is methyl. In some embodiments, R is optionally substituted ethyl. In some embodiments, R is ethyl. In some embodiments, R is −CH2CHF2. In some embodiments, R is −CH2CH2OCH3. In some embodiments, R is optionally substituted propyl. In some embodiments, R is optionally substituted butyl. In some embodiments, R is n-butyl. In some embodiments, R is −(CH2)6NH2. In some embodiments, R is an optionally substituted linear C2-20 aliphatic. In some embodiments, R is optionally substituted linear C2- 20 alkyl. In some embodiments, R is linear C2-20 alkyl. In some embodiments, R is optionally substituted C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 aliphatic. In some embodiments, R is optionally substituted C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl. In some embodiments, R is optionally substituted linear C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl. In some embodiments, R is linear C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is p-methylphenyl. In some embodiments, R is 4-dimethylaminophenyl. In some embodiments, R is 3-pyridinyl. In some embodiments, R In some embodiments, R is benzyl. In someR is optionally substituted 1,3- diazolyl. In some embodiments, R is optionally substituted 2-(1,3)-diazolyl. In some embodiments, R is optionally substituted 1-methyl-2-(1,3)-diazolyl. In some embodiments, R is isopropyl. In some embodiments, R” is −N(R’)2. In some embodiments, R” is −N(CH3)2. In some embodiments, R”, e.g., in −SO2R”, is −OR’, wherein R’ is as described herein. In some embodiments, R’ is R as described herein. In some embodiments, R” is −OCH3. In some embodiments, a linkage is −OP(=O)(−NHSO2R)O−, wherein R is as described herein. In some embodiments, R is optionally substituted linear alkyl as described herein. In some embodiments, R is linear alkyl as described herein. In some embodiments, a linkage is −OP(=O)(−NHSO2CH3)O−. In some embodiments, a linkage is −OP(=O)(−NHSO2CH2CH3)O−. In some embodiments, a linkage is −OP(=O)(−NHSO2CH2CH2OCH3)O−. In some embodiments, a linkage is −OP(=O)(−NHSO2CH2Ph)O−. In some embodiments, a linkage is −OP(=O)(−NHSO2CH2CHF2)O−. In some embodiments, a linkage is Page 72 of 274 12621671v1Attorney Docket No.: 2010581-1497 −OP(=O)(−NHSO2(4-methylphenyl))O−. In some embodiments, −X−RL. In someembodiments, a linkage is −OP(=O)(−X−RL)O−, wherein −X−RL. In some embodiments, a linkage is −OP(=O)(−NHSO2CH(CH3)2)O−. In a linkage is−OP(=O)(−NHSO2N(CH3)2)O−. In some embodiments, a some embodiments, a linkage is n006. In some embodiments, a linkage is n020. In some embodiments, such internucleotidic linkages may be utilized in place of linkages like n001.

[0184] In some embodiments, an internucleotidic linkage has the structure of −P(=O)(−N(R’)C(O)R”)−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=S)(−N(R’)C(O)R”)−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=O)(−N(R’)C(O)R”)O−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=S)(−N(R’)C(O)R”)O−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −OP(=O)(−N(R’)C(O)R”)O−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −OP(=S)(−N(R’)C(O)R”)O−, wherein R” is as described herein. In some embodiments, R’, e.g., of −N(R’)−, is hydrogen or optionally substituted C1-6 aliphatic. In some embodiments, R’ is C1-6 alkyl. In some embodiments, R’ is hydrogen. In some embodiments, R”, e.g., in −C(O)R”, is R’ as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=O)(−NHC(O)R”)−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=S)(−NHC(O)R”)−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=O)(−NHC(O)R”)O−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=S)(−NHC(O)R”)O−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −OP(=O)(−NHC(O)R”)O−, wherein R” is as described herein. In some embodiments, an internucleotidic linkage has the structure of −OP(=S)(−NHC(O)R”)O−, wherein R” is as described herein. In some embodiments, −X−RLis −N(R’)CORL, wherein RLis as described herein. In some embodiments, −X−RLis −N(R’)COR”, wherein R” is as described herein. In some embodiments, −X−RLis −N(R’)COR’, wherein R’ is as described herein. In some embodiments, −X−RLis −NHCOR’, wherein R’ is as described herein. In some embodiments, R’ is R as described herein. In some embodiments, R’ is optionally substituted C1-6 aliphatic. In some embodiments, R’ is optionally substituted C1-6 alkyl. In some embodiments, R’ is optionally substituted phenyl. In some embodiments, R’ is optionally substituted heteroaryl. In some embodiments, R”, e.g., in −C(O)R”, is R. In some embodiments, R is an optionally substituted group selected from C1-6 aliphatic, aryl, heterocyclyl, and heteroaryl. In some embodiments, R is optionally substituted C1-6 aliphatic. In some embodiments, R is Page 73 of 274 12621671v1Attorney Docket No.: 2010581-1497 optionally substituted C1-6alkyl. In some embodiments, R is optionally substituted C1-6alkenyl. In some embodiments, R is optionally substituted C1-6alkynyl. In some embodiments, R is methyl. In some embodiments, −X−RLis −NHC(O)CH3. In some embodiments, R is optionally substituted methyl. In some embodiments, R is −CF3. In some embodiments, R is optionally substituted ethyl. In some embodiments, R is ethyl. In some embodiments, R is −CH2CHF2. In some embodiments, R is −CH2CH2OCH3. In some embodiments, R is optionally substituted C1-20(e.g., C1-6, C2-6, C3-6, C1-10, C2-10, C3-10, C2-20, C3-20, C10-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) aliphatic. In some embodiments, R is optionally substituted C1-20 (e.g., C1-6, C2-6, C3-6, C1-10, C2-10, C3-10, C2-20, C3-20, C10-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) alkyl. In some embodiments, R is an optionally substituted linear C2-20 aliphatic. In some embodiments, R is optionally substituted linear C2-20 alkyl. In some embodiments, R is linear C2-20 alkyl. In some embodiments, R is optionally substituted C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 aliphatic. In some embodiments, R is optionally substituted C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl. In some embodiments, R is optionally substituted linear C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl. In some embodiments, R is linear C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl. In some embodiments, R is optionally substituted aryl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is p-methylphenyl. In some embodiments, R is benzyl. In some embodiments, R is optionally substituted heteroaryl. In some embodiments, R is optionally substituted 1,3-diazolyl. In some embodiments, R is optionally substituted 2-(1,3)-diazolyl. In some embodiments, R is optionally substituted 1-methyl-2-(1,3)-diazolyl. In some embodiments, RLis −(CH2)5NH2. In some embodiments, . In some embodiments, RL. In some embodiments,embodiments, R” is −N(CH3)−X−RLis −N(R’)CON(RL)2, wherein each of R’ and RLis independently as described herein. In some embodiments, −X−RLis −NHCON(RL)2, wherein RLis as described herein. In some embodiments, two R’ or two RLare taken together with the nitrogen atom to which they are attached to form a ring as described herein, e.g., optionally , inIn some embodiments, is optionally substituted C1-6aliphatic. In some embodiments, is optionally substituted C1-6alkyl. In some embodiments, R” is −OCH3. In some embodiments, −X−RLis −N(R’)C(O)ORL, wherein each Page 74 of 274 12621671v1Attorney Docket No.: 2010581-1497 of R’ and RLis independently as described herein. In some embodiments, R . In some embodiments, −X−RLis −NHC(O)OCH3. In some embodiments, −X−RL2. In someembodiments, a linkage is −OP(O)(NHC(O)CH3)O−. In some linkage is −OP(O)(NHC(O)OCH3)O−. In some embodiments, a linkage is −OP(O)(NHC(O)(p-methylphenyl))O−. In some embodiments, a linkage is −OP(O)(NHC(O)N(CH3)2)O−. In some embodiments, −X−RLis −N(R’)RL, wherein each of R’ and RLis independently as described herein. In some embodiments, −X−RLis −N(R’)RL, wherein each of R’ and RLis independently not hydrogen. In some embodiments, −X−RLis −NHRL, wherein RLis as described herein. In some embodiments, RLis not hydrogen. In some embodiments, RLis optionally substituted aryl or heteroaryl. In some embodiments, RLis optionally substituted aryl. In some embodiments, RLis optionally substituted phenyl. In some embodiments, −X−RLis −N(R’)2, wherein each R’ is independently as described herein. In some embodiments, −X−RLis −NHR’, wherein R’ is as described herein. In some embodiments, −X−RLis −NHR, wherein R is as described herein. In some embodiments, −X−RLis RL, wherein RLis as described herein. In some embodiments, RLis −N(R’)2, wherein each R’ is independently as described herein. In some embodiments, RLis −NHR’, wherein R’ is as described herein. In some embodiments, RLis −NHR, wherein R is as described herein. In some embodiments, RLis −N(R’)2, wherein each R’ is independently as described herein. In some embodiments, none of R’ in −N(R’)2is hydrogen. In some embodiments, RLis −N(R’)2, wherein each R’ is independently C1-6aliphatic. In some embodiments, RLis −L−R’, wherein each of L and R’ is independently as described herein. In some embodiments, RLis −L−R, wherein each of L and R is independently as described herein. In some embodiments, RLis −N(R’)−Cy−N(R’)−R’. In some embodiments, RLis −N(R’)−Cy−C(O)−R’. In some embodiments, RLis −N(R’)−Cy−O−R’. In some embodiments, RLis −N(R’)−Cy−SO2−R’. In some embodiments, RLis −N(R’)−Cy−SO2−N(R’)2. In some embodiments, RLis −N(R’)−Cy−C(O)−N(R’)2. In some embodiments, RLis −N(R’)−Cy−OP(O)(R”)2. In some embodiments, −Cy− is an optionally substituted bivalent aryl group. In some embodiments, −Cy− is optionally substituted phenylene. In some embodiments, −Cy− is optionally substituted 1,4-phenylene. In some embodiments, −Cy− is 1,4-phenylene. In some embodiments, RLis −N(CH3)2. In some embodiments, RLis −N(i-Pr)2. In some embodiments, RL. Insome embodiments, RL. In someembodiments, RL. In some embodiments, RL. In somePage 75 of 274 12621671v1Attorney Docket No.: 2010581-1497 embodiments, RLis . In some embodiments, RL. In someembodiments, RL. In some. In some embodiments, RLis . In some embodiments, RLis. In someembodiments, RLis . In some embodiments, RL. Insome embodiments, RL. In some embodiments, RL. Insome embodiments, RLis . In some embodiments, RL. In, , ,Page 76 of 274 12621671v1Attorney Docket No.: 2010581-1497 , ,RLis −N(R’)−C(O)−Cy−O−R’. In some embodiments, RLis −N(R’)−C(O)−Cy−R’. In some embodiments, RLis −N(R’)−C(O)−Cy−C(O)−R’. In some embodiments, RLis −N(R’)−C(O)−Cy−N(R’)2. In some embodiments, RLis −N(R’)−C(O)−Cy−SO2−N(R’)2. In some embodiments, RLis −N(R’)−C(O)−Cy−C(O)−N(R’)2. In some embodiments, RLis −N(R’)−C(O)−Cy−C(O)−N(R’)−SO2−R’. In , , , , ,12621671v1Attorney Docket No.: 2010581-1497 , ,−, −, −, −SO2N(R’)−, or −Cy−. In some embodiments, a methylene unit is replaced with −Cy−. In some embodiments, −Cy− is an optionally substituted bivalent aryl group. In some embodiments, −Cy− is optionally substituted phenylene. In some embodiments, −Cy− is optionally substituted 1,4-phenylene. In some embodiments, −Cy− is an optionally substituted bivalent 5-20 (e.g.5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) membered heteroaryl group having 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) heteroatoms. In some embodiments, −Cy− is monocyclic. In some embodiments, −Cy− is bicyclic. In some embodiments, −Cy− is polycyclic. In some embodiments, each monocyclic unit in −Cy− is independently 3-10 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) membered, and is independently saturated, partially saturated, or aromatic. In some embodiments, −Cy− is an optionally substituted 3-20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) membered monocyclic, bicyclic or polycyclic aliphatic group. In some embodiments, −Cy− is an optionally substituted 3-20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) membered monocyclic, bicyclic or polycyclic heteroaliphatic group having 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) heteroatoms.

[0186] In some embodiments, an internucleotidic linkage has the structure of −P(=O)(−N(R’)P(O)(R”)2)−, wherein each R” is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=S)(−N(R’)P(O)(R”)2)−, wherein each R” is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=O)(−N(R’)P(O)(R”)2)O−, wherein each R” is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=S)(−N(R’)P(O)(R”)2)O−, wherein each R” is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −OP(=O)(−N(R’)P(O)(R”)2)O−, wherein each R” is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −OP(=S)(−N(R’)P(O)(R”)2)O−, wherein each R” is independently as described herein. In some embodiments, R’, e.g., of −N(R’)−, is hydrogen or optionally substituted C1-6aliphatic. In some embodiments, R’ is C1-6alkyl. In some embodiments, R’ is hydrogen. In some embodiments, R”, e.g., in −P(O)(R”)2, is R’ as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=O)(−NHP(O)(R”)2)−, wherein each Page 78 of 274 12621671v1Attorney Docket No.: 2010581-1497 R” is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=S)(−NHP(O)(R”)2)−, wherein each R” is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=O)(−NHP(O)(R”)2)O−, wherein each R” is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −P(=S)(−NHP(O)(R”)2)O−, wherein each R” is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −OP(=O)(−NHP(O)(R”)2)O−, wherein each R” is independently as described herein. In some embodiments, an internucleotidic linkage has the structure of −OP(=S)(−NHP(O)(R”)2)O−, wherein each R” is independently as described herein. In some embodiments, an occurrence of R”, e.g., in −P(O)(R”)2, is R. In some embodiments, R is an optionally substituted group selected from C1-6 aliphatic, aryl, heterocyclyl, and heteroaryl. In some embodiments, R is optionally substituted C1-6 aliphatic. In some embodiments, R is optionally substituted C1-6 alkyl. In some embodiments, R is optionally substituted C1-6 alkenyl. In some embodiments, R is optionally substituted C1-6 alkynyl. In some embodiments, R is methyl. In some embodiments, R is optionally substituted methyl. In some embodiments, R is −CF3. In some embodiments, R is optionally substituted ethyl. In some embodiments, R is ethyl. In some embodiments, R is −CH2CHF2. In some embodiments, R is −CH2CH2OCH3. In some embodiments, R is optionally substituted C1-20 (e.g., C1-6, C2-6, C3-6, C1-10, C2-10, C3-10, C2-20, C3-20, C10-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) aliphatic. In some embodiments, R is optionally substituted C1-20 (e.g., C1-6, C2-6, C3-6, C1-10, C2-10, C3-10, C2-20, C3-20, C10-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) alkyl. In some embodiments, R is an optionally substituted linear C2-20 aliphatic. In some embodiments, R is optionally substituted linear C2-20 alkyl. In some embodiments, R is linear C2-20 alkyl. In some embodiments, R is isopropyl. In some embodiments, R is optionally substituted C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 aliphatic. In some embodiments, R is optionally substituted C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl. In some embodiments, R is optionally substituted linear C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl. In some embodiments, R is linear C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl. In some embodiments, each R” is independently R as described herein, for example, in some embodiments, each R” is methyl. In some embodiments, R” is optionally substituted aryl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is p- methylphenyl. In some embodiments, R is benzyl. In some embodiments, R is optionally substituted heteroaryl. In some embodiments, R is optionally substituted 1,3-diazolyl. In some embodiments, R is optionally substituted 2-(1,3)-diazolyl. In some embodiments, R is optionally substituted 1-methyl-2-(1,3)- diazolyl. In some embodiments, an occurrence of R” is −N(R’)2. In some embodiments, R” is −N(CH3)2. In some embodiments, an occurrence of R”, e.g., in −P(O)(R”)2, is −OR’, wherein R’ is as described herein. In some embodiments, R’ is R as described herein. In some embodiments, is optionally substituted C1-6aliphatic. In some embodiments, is optionally substituted C1-6alkyl. In some embodiments, R” is −OCH3. In some embodiments, each R” is −OR’ as described herein. In some embodiments, each R” is −OCH3. In some Page 79 of 274 12621671v1Attorney Docket No.: 2010581-1497 embodiments, each R” is −OH. In some embodiments, a linkage is −OP(O)(NHP(O)(OH)2)O−. In some embodiments, a linkage is −OP(O)(NHP(O)(OCH3)2)O−. In some embodiments, a linkage is −OP(O)(NHP(O)(CH3)2)O−.

[0187] In some embodiments, −N(R”)2is −N(R’)2. In some embodiments, −N(R”)2is −NHR. In some embodiments, −N(R”)2 is −NHC(O)R. In some embodiments, −N(R”)2 is −NHC(O)OR. In some embodiments, −N(R”)2is −NHS(O)2R.

[0188] In some embodiments, an internucleotidic linkage is a phosphoryl guanidine internucleotidic linkage. In some embodiments, an internucleotidic linkage comprises −X−RLas described herein. In some embodiments, −X−RLis −N=C(–LL–RL)2. In some embodiments, −X−RLis −N=C[N(RL)2]2. In some embodiments, −X−RLis −N=C[NR’RL]2. In some embodiments, −X−RLis −N=C[N(R’)2]2. In some embodiments, −X−RLis −N=C[N(RL)2](CHRL1RL2), wherein each of RL1and RL2is independently as described herein. In some embodiments, −X−RLis −N=C(NR’RL)(CHRL1RL2), wherein each of RL1and RL2is independently as described herein. In some embodiments, −X−RLis −N=C(NR’RL)(CR’RL1RL2), wherein each of RL1and RL2is independently as described herein. In some embodiments, −X−RLis −N=C[N(R’)2](CHR’RL2). In some embodiments, −X−RLis −N=C[N(RL)2](RL). In some embodiments, −X−RLis −N=C(NR’RL)(RL). In some embodiments, −X−RLis −N=C(NR’RL)(R’). In some embodiments, −X−RLis −N=C[N(R’)2](R’). In some embodiments, −X−RLis −N=C(NR’RL1)(NR’RL2), wherein each RL1and RL2is independently RL, and each R’ and RLis independently as described herein. In some embodiments, −X−RLis −N=C(NR’RL1)(NR’RL2), wherein variable is independently as described herein. In some embodiments, −X−RLis −N=C(NR’RL1)(CHR’RL2), wherein variable is independently as described herein. In some embodiments, −X−RLis −N=C(NR’RL1)(R’), wherein variable is independently as described herein. In some embodiments, each R’ is independently R. In some embodiments, R is optionally substituted C1-6 aliphatic. In some embodiments, R is methyl. In some . In some embodiments, two groups selected from R’, RL, RL1, RL2, etc. (in someatom (e.g., −N(R’)2, or −NR’RL, or −N(RL)2, wherein R’ and RLcan independently be R as described herein), etc.), or on different atoms (e.g., the two R’ in −N=C(NR’RL)(CR’RL1RL2) or −N=C(NR’RL1)(NR’RL2); can also be two other variables that can be R, e.g., RL, RL1, RL2, etc.)) are independently R and are taken together with their intervening atoms to form a ring as described herein. In some embodiments, two of R, R’, RL, RL1, or RL2on the same atom, e.g., of −N(R’)2, −N(RL)2, −NR’RL, −NR’RL1, −NR’RL2, −CR’RL1RL2, etc., are taken together to form a ring as described herein. In some embodiments, two R’, RL, RL1, or RL2on two different atoms, e.g., the two R’ in −N=C(NR’RL)(CR’RL1RL2), −N=C(NR’RL1)(NR’RL2), etc. are taken together to form a ring as described herein. In some embodiments, a formed ring is an optionally substituted 3-20 (e.g., 3-15, 3-12, 3-10, 3-9, 3-8, 3-7, 3-6, 4-15, 4-12, 4-10, 4-9, 4-8, 4-7, 4-6, 5-15, 5-12, 5-10, 5-9, 5-8, 5-7, 5-6, 1, 2, 3, 4, 5, 6, 7, 8, Page 80 of 274 12621671v1Attorney Docket No.: 2010581-1497 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) monocyclic, bicyclic or tricyclic ring having 0-5 additional heteroatoms. In some embodiments, a formed ring is monocyclic as described herein. In some embodiments, a formed ring is an optionally substituted 5-10 membered monocyclic ring. In some embodiments, a formed ring is bicyclic. In some embodiments, a formed ring is polycyclic. In some embodiments, two groups that are or can be R (e.g., the two R’ in −N=C(NR’RL)(CR’RL1RL2) or −N=C(NR’RL1)(NR’RL2), the two R’ in −N=C(NR’RL)(CR’RL1RL2), −N=C(NR’RL1)(NR’RL2), etc.) are taken together to form an optionally substituted bivalent hydrocarbon chain, e.g., an optionally substituted C1-20aliphatic chain, optionally substituted −(CH2)n− wherein n is 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, a hydrocarbon chain is saturated. In some embodiments, a hydrocarbon chain is partially unsaturated. In some embodiments, a hydrocarbon chain is unsaturated. In some embodiments, two groups that are or can be R (e.g., the two R’ in −N=C(NR’RL)(CR’RL1RL2) or −N=C(NR’RL1)(NR’RL2), the two R’ in −N=C(NR’RL)(CR’RL1RL2), −N=C(NR’RL1)(NR’RL2), etc.) are taken together to form an optionally substituted bivalent heteroaliphatic chain, e.g., an optionally substituted C1-20 heteroaliphatic chain having 1-10 heteroatoms. In some embodiments, a heteroaliphatic chain is saturated. In some embodiments, a heteroaliphatic chain is partially unsaturated. In some embodiments, a heteroaliphatic chain is unsaturated. In some embodiments, a chain is optionally substituted −(CH2)−. In some embodiments, a chain is optionally substituted −(CH2)2−. In some embodiments, a chain is optionally substituted −(CH2)−. In some embodiments, a chain is optionally substituted −(CH2)2−. In some embodiments, a chain is optionally substituted −(CH2)3−. In some embodiments, a chain is optionally substituted −(CH2)4−. In some embodiments, a chain is optionally substituted −(CH2)5−. In some embodiments, a chain is optionally substituted −(CH2)6−. In some embodiments, a chain is optionally substituted −CH=CH−. In some embodiments, a chain is optionally. In some embodiments, a chain is optionally . In some embodiments, a chain is optionallysubstituted . In some embodiments, a chain is optionally . In some embodiments,a chain is optionally . In some embodiments, a chain is optionally .In some embodiments, a chain is optionally . In some embodiments, a chain is optionallyPage 81 of 274 12621671v1Attorney Docket No.: 2010581-1497 substituted . In some as describedherein. For examples, in some . In some embodiments, −X−RLis. In some embodiments, −X−RLis . In some embodiments, −X−RLis some . In some embodiments, −X−RLissome . In some embodiments, −X−RLissome . In some embodiments, −X−RLisIn some embodiments, −N(R’)2, −N(R)2, −N(RL)2, −NR’RL, −NR’RL1, −NR’RL2,−NRL1RL2, etc. is a formed ring. In some embodiments, a ring is optionally . In someembodiments, a ring is optionally . In some embodiments, a ring is optionally substituted. In some embodiments, a ring is optionally . In some embodiments, a ring isPage 82 of 274 12621671v1Attorney Docket No.: 2010581-1497 optionally substituted . In some embodiments, a ring is optionally substituted . Insome embodiments, substituted . In somesubstituted . In some a substituted . In some. In some embodiments, a ring is optionally . In some embodiments, a ring isoptionally . In some embodiments, a ring is optionally . In someembodiments, a ring is optionally .

[0189] In some embodiments, RL1andL1 L2same. In some embodiments, R and R are different. In some embodiments, each of RL1and RL2is independently RLas described herein, e.g., below.

[0190] In some embodiments, RLis optionally substituted C1-30 aliphatic. In some embodiments, RLis optionally substituted C1-30 alkyl. In some embodiments, RLis linear. In some embodiments, RLis optionally substituted linear C1-30 alkyl. In some embodiments, RLis optionally substituted C1-6 alkyl. In some embodiments, RLis methyl. In some embodiments, RLis ethyl. In some embodiments, RLis n-propyl. In some embodiments, RLis isopropyl. In some embodiments, RLis n-butyl. In some embodiments, RLis tert-butyl. In some embodiments, RLis (E)−CH2−CH=CH−CH2−CH3. In some embodiments, RLis (Z)−CH2−CH=CH−CH2−CH3. In some embodiments, RL. In someembodiments, RLis . In some embodiments, RLis CH3(CH2)2C≡CC≡ C(CH2)3−. In some5C≡C−. In some embodiments, RLoptionally substituted aryl. In some embodiments, RLis optionally substituted phenyl. In some embodiments, RLis phenyl substituted with one or more halogen. In some embodiments, RLis phenyl optionally substituted with halogen, −N(R’), or −N(R’)C(O)R’. In some embodiments, RLis phenyl optionally substituted with −Cl, −Br, −F, −N(Me)2, or −NHCOCH3. In some embodiments, RLis −LL−R’, wherein LLis an optionally substituted C1-20saturated, partially unsaturated or unsaturated hydrocarbon chain. In some embodiments, such a hydrocarbon chain is Page 83 of 274 12621671v1Attorney Docket No.: 2010581-1497 linear. In some embodiments, such a hydrocarbon chain is unsubstituted. In some embodiments, LLis (E)−CH2−CH=CH−. In some embodiments, LLis −CH2−C≡C−CH2−. In some embodiments, LLis −(CH2)3−. In some embodiments, LLis −(CH2)4−. In some embodiments, LLis −(CH2)n−, wherein n is 1-30 (e.g., 1-20, 5- 30, 6-30, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, etc.). In some embodiments, R’ is optionally substituted aryl as described herein. In some embodiments, R’ is optionally substituted phenyl. In some embodiments, R’ is phenyl. In some embodiments, R’ is optionally substituted heteroaryl as described herein. In some embodiments, R’ is 2’-pyridinyl. In some embodiments, R’ is 3’-pyridinyl. In some embodiments, RL. In some embodiments,. In some embodiments, RL. In some embodiments, RLis −LL–N(R’)2, wherein each variable is independently as embodiments, each R’ is independently C1-6aliphatic as described herein. In some embodiments, −N(R’)2 is −N(CH3)2. In some embodiments, −N(R’)2 is −NH2. In some embodiments, RLis −(CH2)n−N(R’)2, wherein n is 1-30 (e.g., 1-20, 5-30, 6-30, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, etc.). In some embodiments, RLis −(CH2CH2O)n−CH2CH2−N(R’)2, wherein n is 1-30 (e.g., 1-20, 5-30, 6-30, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, etc.). In some embodiments, RLis . In some embodiments, RLis . In some embodiments, RLis. In some embodiments, RLis −(CH2)n−NH2. In some embodiments, RLisIn some embodiments, RLis −(CH2CH2O)n−CH2CH2−R’, wherein n is 1-30 (e.g., 1-20, 5-30, 6-30, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, etc.). In some embodiments, RLis −(CH2CH2O)n−CH2CH2CH3, wherein n is 1-30 (e.g., 1-20, 5-30, 6-30, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, etc.). In some embodiments, RLis −(CH2CH2O)n−CH2CH2OH, wherein n is 1-30 (e.g., 1-20, 5-30, 6-30, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, etc.). In some embodiments, RLis or comprises a carbohydrate moiety, e.g., GalNAc. In some embodiments, RLis −LL−GalNAc. In some . In some embodiments, one or more methyleneoptionally substituted 1,4-phenylene, a 3-30 membered bivalent optionally substituted monocyclic, bicyclic, or polycyclic cycloaliphatic ring, etc.), −O−, −N(R’)− (e.g., −NH), −C(O)−, −C(O)N(R’)− (e.g., −C(O)NH−), −C(NR’)− (e.g., −C(NH)−), −N(R’)C(O)(N(R’)− (e.g., −NHC(O)NH−), −N(R’)C(NR’)(N(R’)− (e.g., −NHC(NH)NH−), Page 84 of 274 12621671v1Attorney Docket No.: 2010581-1497 −(CH2CH2O)n−, etc. For example, in some Inis is is isoptionally substituted connected through a linker (which can be bivalent or polyvalent). For example, in some embodiments, RLis ,wherein n is 0-20. In some embodiments, RL, wherein n is 0-20. In some embodiments, RLis R’ as described herein. Ascan independently be R’. In some embodiments, R’ is R as described herein. As described herein, various variables can independently be R. In some embodiments, R is optionally substituted C1-6 aliphatic. In some embodiments, R is optionally substituted C1-6alkyl. In some embodiments, R is methyl. In some embodiments, R is optionally substituted Page 85 of 274 12621671v1Attorney Docket No.: 2010581-1497 cycloaliphatic. In some embodiments, R is optionally substituted cycloalkyl. In some embodiments, R is optionally substituted aryl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is optionally substituted heteroaryl. In some embodiments, R is optionally substituted heterocyclyl. In some embodiments, R is optionally substituted C1-20heterocyclyl having 1-5 heteroatoms, e.g., one of which is nitrogen. In some embodiments, R is optionally substituted . In some embodiments, R is optionally substituted . In some embodiments, R is optionally substituted . In some embodiments, R isoptionally . In some embodiments, R is . In someembodiments, R is optionally . In some embodiments, R is optionally substituted. In some embodiments, R is optionally . In some embodiments, R isoptionally . In some embodiments, R is optionally . In someembodiments, R is optionally In some embodiments, R is optionally. In some embodiments, R is optionally . In some embodiments, R is optionally. In some embodiments, R is optionally .

[0191] In some In some .In some . In some . In somePage 86 of 274 12621671v1Attorney Docket No.: 2010581-1497 embodiments, . In some . In some. In some embodiments, −X−RLis, ,Page 87 of 274 12621671v1Attorney Docket No.: 2010581-1497 .In some embodiments, −X−RL is. In some embodiments, −X−RLLsome as In some embodiments, R as herein.

[0193] In some embodiments, R” or RLis or comprises an additional chemical moiety. In some embodiments, R” or RLis or comprises an additional chemical moiety, wherein the additional chemical moiety is or comprises a carbohydrate moiety. In some embodiments, R” or RLis or comprises a GalNAc. In some embodiments, RLor R” is replaced with, or is utilized to connect to, an additional chemical moiety.

[0194] In some embodiments, X is –O–. In some embodiments, X is –S–. In some embodiments, X is −LL−N(–LL–RL)−LL−. In some embodiments, X is −N(–LL–RL)−LL−. In some embodiments, X is −LL−N(– LL–RL)−. In some embodiments, X is −N(–LL–RL)−. In some embodiments, X is −LL−N=C(–LL–RL)−LL−. In some embodiments, X is −N=C(–LL–RL)−LL−. In some embodiments, X is −LL−N=C(–LL–RL)−. In some embodiments, X is −N=C(–LL–RL)−. In some embodiments, X is LL. In some embodiments, X is a covalent bond.

[0195] In some embodiments, Y is a covalent bond. In some embodiments, Y is −O−. In some embodiments, Y is −N(R’)−. In some embodiments, Z is a covalent bond. In some embodiments, Z is −O−. In some embodiments, Z is −N(R’)−. In some embodiments, R’ is R. In some embodiments, R is −H. In some embodiments, R is optionally substituted C1-6 aliphatic. In some embodiments, R is methyl. In some embodiments, R is ethyl. In some embodiments, R is propyl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is phenyl.

[0196] As described herein, various variables in structures in the present disclosure can be or comprise R. Suitable embodiments for R are described extensively in the present disclosure. As appreciated by those skilled in the art, R embodiments described for a variable that can be R may also be applicable to another variable that can be R. Similarly, embodiments described for a component / moiety (e.g., L) for a variable may also be applicable to other variables that can be or comprise the component / moiety.

[0197] In some embodiments, R” is R’. In some embodiments, R” is −N(R’)2.

[0198] In some embodiments, −X−RLis −SH. In some embodiments, −X−RLis −OH.

[0199] In some embodiments, −X−RLis −N(R’)2. In some embodiments, each R’ is independently optionally substituted C1-6aliphatic. In some embodiments, each R’ is independently methyl.

[0200] In some embodiments, a non-negatively charged internucleotidic linkage has the structure of Page 88 of 274 12621671v1Attorney Docket No.: 2010581-1497 −OP(=O)(−N=C((N(R’)2)2−O−. In some embodiments, a R’ group of one N(R’)2is R, a R’ group of the other N(R’)2is R, and the two R groups are taken together with their intervening atoms to form an optionally substituted ring, e.g., a 5-membered ring as in n001. In some embodiments, each R’ is independently R, wherein each R is independently optionally substituted C1-6aliphatic.

[0201] In some embodiments, −X−RLis −N=C(–LL–R’)2. In some embodiments, −X−RLis −N=C(– LL1−LL2−LL3–R’)2, wherein each LL1, LL2and LL3is independently L”, wherein each L” is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C1-10aliphatic group and a C1-10 heteroaliphatic group having 1-5 heteroatoms, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C1-6 alkylene, C1-6 alkenylene, , a bivalent C1–C6heteroaliphatic group having 1-5 heteroatoms, −C(R’)2−, −Cy−, −O−, −S−, −S−S−, −N(R’)−, −C(O)−, −C(S)−, −C(NR’)−, −C(O)N(R’)−, −N(R’)C(O)N(R’)−, −N(R’)C(O)O−, −S(O)−, −S(O)2−, −S(O)2N(R’)−, −C(O)S−, −C(O)O−, −P(O)(OR’)−, −P(O)(SR’)−, −P(O)(R’)−, −P(O)(NR’)−, −P(S)(OR’)−, −P(S)(SR’)−, −P(S)(R’)−, −P(S)(NR’)−, −P(R’)−, −P(OR’)−, −P(SR’)−, −P(NR’)−, −P(OR’)[B(R’)3]−, −OP(O)(OR’)O−, −OP(O)(SR’)O−, −OP(O)(R’)O−, −OP(O)(NR’)O−, −OP(OR’)O−, −OP(SR’)O−, −OP(NR’)O−, −OP(R’)O−, or −OP(OR’)[B(R’)3]O−, and one or more nitrogen or carbon atoms are optionally and independently replaced with CyL. In some embodiments, LL2is −Cy−. In some embodiments, LL1is a covalent bond. In some embodiments, LL3is a covalent bond. In some embodiments, −X−RLis −N=C(–LL1−Cy−LL3–R’)2. In someembodiments, . In someembodiments, . In some . In somePage 89 of 274 12621671v1Attorney Docket No.: 2010581-1497 .

[0202] utilized in the present disclosure, L is covalent bond. In somesubstituted, linear or branched group selected from a C1-30 aliphatic group and a C1-30heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C1-6alkylene, C1-6alkenylene, , a bivalent C1–C6 heteroaliphatic group having 1-5 heteroatoms, −C(R’)2−, −Cy−, −O−, −S−, −S−S−, −N(R’)−, −C(O)−, −C(S)−, −C(NR’)−, −C(O)N(R’)−, −N(R’)C(O)N(R’)−, −N(R’)C(O)O−, −S(O)−, −S(O)2−, −S(O)2N(R’)−, −C(O)S−, −C(O)O−, −P(O)(OR’)−, −P(O)(SR’)−, −P(O)(R’)−, −P(O)(NR’)−, −P(S)(OR’)−, −P(S)(SR’)−, −P(S)(R’)−, −P(S)(NR’)−, −P(R’)−, −P(OR’)−, −P(SR’)−, −P(NR’)−, −P(OR’)[B(R’)3]−, −OP(O)(OR’)O−, −OP(O)(SR’)O−, −OP(O)(R’)O−, −OP(O)(NR’)O−, −OP(OR’)O−, −OP(SR’)O−, −OP(NR’)O−, −OP(R’)O−, or −OP(OR’)[B(R’)3]O−, and one or more nitrogen or carbon atoms are optionally and independently replaced with CyL. In some embodiments, L is a bivalent, optionally substituted, linear or branched group selected from a C1-30aliphatic group and a C1-30heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from , −C(R’)2−, −Cy−, −O−, −S−, −S−S−, −N(R’)−, −C(O)−, −C(S)−, −C(NR’)−, −C(O)N(R’)−,(O)N(R’)−, −N(R’)C(O)O−, −S(O)−, −S(O)2−, −S(O)2N(R’)−, −C(O)S−, −C(O)O−, −P(O)(OR’)−, −P(O)(SR’)−, −P(O)(R’)−, −P(O)(NR’)−, −P(S)(OR’)−, −P(S)(SR’)−, −P(S)(R’)−, −P(S)(NR’)−, −P(R’)−, −P(OR’)−, −P(SR’)−, −P(NR’)−, −P(OR’)[B(R’)3]−, −OP(O)(OR’)O−, −OP(O)(SR’)O−, −OP(O)(R’)O−, −OP(O)(NR’)O−, −OP(OR’)O−, −OP(SR’)O−, −OP(NR’)O−, −OP(R’)O−, or −OP(OR’)[B(R’)3]O−, and one or more nitrogen or carbon atoms are optionally and independently replaced with CyL. In some embodiments, L is a bivalent, optionally substituted, linear or branched group selected from a C1-10aliphatic group and a C1-10heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from , −C(R’)2−, −Cy−, −O−, −S−, −S−S−, −N(R’)−, −C(O)−, −C(S)−, −C(NR’)−, −C(O)N(R’)−, −N(R’)C(O)N(R’)−, −N(R’)C(O)O−, −S(O)−, −S(O)2−, −S(O)2N(R’)−, −C(O)S−, −C(O)O−, −P(O)(OR’)−, −P(O)(SR’)−, −P(O)(R’)−, −P(O)(NR’)−, −P(S)(OR’)−, −P(S)(SR’)−, −P(S)(R’)−, −P(S)(NR’)−, −P(R’)−, −P(OR’)−, −P(SR’)−, −P(NR’)−, −P(OR’)[B(R’)3]−, −OP(O)(OR’)O−, −OP(O)(SR’)O−, −OP(O)(R’)O−, −OP(O)(NR’)O−, −OP(OR’)O−, −OP(SR’)O−, −OP(NR’)O−, −OP(R’)O−, or −OP(OR’)[B(R’)3]O−, and one or more nitrogen or carbon atoms are optionally and independently replaced with CyL. In some embodiments, one or more methylene units are optionally and independently replaced by an optionally substituted group selected from , −C(R’)2−, −Cy−, −O−, −S−, −S−S−, −N(R’)−, −C(O)−, −C(S)−, −C(NR’)−, −C(O)N(R’)−, −N(R’)C(O)N(R’)−, −N(R’)C(O)O−, −S(O)−, −S(O)2−, −S(O)2N(R’)−, Page 90 of 274 12621671v1Attorney Docket No.: 2010581-1497 −C(O)S−, or −C(O)O−.

[0203] In some embodiments, an internucleotidic linkage is a phosphoryl guanidine internucleotidic linkage. In some embodiments, −X−RLis −N=C[N(R’)2]2.In some embodiments, each R’ is independently R. In some embodiments, R is optionally substituted C1-6aliphatic. In some embodiments, R is methyl. In some some embodiments, one R’ on a nitrogen atom is taken with a R’ on the other herein.

[0204] In some , wherein R1and R2are independently R’. In someembodiments, −X−RLis . In some embodiments, −X−RLis . In some embodiments, two R’ on the same nitrogen are taken together to form a ring as described herein. In some embodiments, is is isPage 91 of 274 12621671v1Attorney Docket No.: 2010581-1497 isIn some embodiments, R is optionally substituted C1-6aliphatic. In some embodiments, R is optionally substituted C1-6alkyl. In some embodiments, R is methyl.

[0206] In some embodiments, −X−RLis selected from Tables below. In some embodiments, X is as described herein. In some embodiments, RLis as described herein. In some embodiments, a linkage has the structure of −Y−PL(−X−RL)−Z−, wherein −X−RLis selected from Tables below, and each other variable is independently as described herein. In some embodiments, a linkage has the structure of or comprises −P(O)(−X−RL)−, wherein −X−RLis selected from Tables below. In some embodiments, a linkage has the structure of or comprises −P(S)(−X−RL)−, wherein −X−RLis selected from Tables below. In some embodiments, a linkage has the structure of or comprises −P(−X−RL)−, wherein −X−RLis selected from Tables below. In some embodiments, a linkage has the structure of or comprises −P(O)(−X−RL)−O−, wherein −X−RLis selected from Tables below. In some embodiments, a linkage has the structure of or comprises −P(S)(−X−RL)−O−, wherein −X−RLis selected from Tables below. In some embodiments, a linkage has the structure of or comprises −P(−X−RL)−O−, wherein −X−RLis selected from Tables below. In some embodiments, a linkage has the structure of −P(O)(−X−RL)−O−, wherein −X−RLis selected from Tables below. In some embodiments, a linkage has the structure of −P(S)(−X−RL)−O−, wherein −X−RLis selected from Tables below. In some embodiments, a linkage has the structure of −P(−X−RL)−O−, wherein −X−RLis selected from Tables below. In some embodiments, P is bonded to a nitrogen atom (e.g., a nitrogen atom in sm01, sm18, etc.). In some embodiments, a linkage has the structure of or comprises −O−P(O)(−X−RL)−O−, wherein −X−RLis selected from Tables below. In some embodiments, a linkage has the structure of or comprises −O−P(S)(−X−RL)−O−, wherein −X−RLis selected from Tables below. In some embodiments, a linkage has the structure of or comprises −O−P(−X−RL)−O−, wherein −X−RLis selected from Tables below. In some embodiments, a linkage has the structure of −O−P(O)(−X−RL)−O−, wherein −X−RLis selected from Tables below. In some embodiments, a linkage has the structure of −O−P(S)(−X−RL)−O−, wherein −X−RLis selected from Tables below. In some embodiments, a linkage has the structure of −O−P(−X−RL)−O−, wherein −X−RLis selected from Tables below. In some embodiments, the Tables below, n is 0-20 or as described herein. As those skilled in the art appreciate, a linkage may exist in a salt form. Page 92 of 274 12621671v1Attorney Docket No.: 2010581-1497 Table L-1. Certain useful moieties bonded to linkage phosphorus (e.g., −X−RL).Page 93 of 274 12621671v1Attorney Docket No.: 2010581-1497Page 94 of 274 12621671v1Attorney Docket No.: 2010581-1497 ,(Me)2, Page 95 of 274 12621671v1Attorney Docket No.: 2010581-1497 or −NHCOCH3. Table L-2. Certain useful moieties bonded to linkage phosphorus (e.g., −X−RL).Page 96 of 274 12621671v1Attorney Docket No.: 2010581-1497 Table L-4. Certain useful moieties bonded to linkage phosphorus (e.g., −X−RL).Page 97 of 274 12621671v1Attorney Docket No.: 2010581-1497Page 98 of 274 12621671v1Attorney Docket No.: 2010581-1497Page 99 of 274 12621671v1Attorney Docket No.: 2010581-1497linkage or a neutral internucleotidic linkage, has the structure of −LL1−CyIL−LL2−. In some embodiments, LL1Page 100 of 274 12621671v1Attorney Docket No.: 2010581-1497 is bonded to a 3’-carbon of a sugar. In some embodiments, LL2is bonded to a 5’-carbon of a sugar. In some embodiments, LL1is −O−CH2−. In some embodiments, LL2is a covalent bond. In some embodiments, LL2is a −N(R’)−. In some embodiments, LL2is a −NH−. In some embodiments, LL2is bonded to a 5’-carbon of a sugar, which 5’-carbon is substituted with =O. In some embodiments, CyILis optionally substituted 3-10 membered saturated, partially unsaturated, or aromatic ring having 0-5 heteroatoms. In some embodiments, CyILis an optionally substituted triazole ring. In some embodiments, CyILis . In someembodiments, a linkage is .

[0208] In some negatively charged internucleotidic linkage has the structure of−OP(=W)(−N(R’)2)

[0209] In some embodiments, R’ is R. In some embodiments, R’ is −H. In some embodiments, R’ is −C(O)R. In some embodiments, R’ is −C(O)OR. In some embodiments, R’ is −S(O)2R.

[0210] In some embodiments, R” is −NHR’. In some embodiments, −N(R’)2is −NHR’.

[0211] As described herein, some embodiments, R is −H. In some embodiments, R is optionally substituted C1-6aliphatic. In some embodiments, R is optionally substituted C1-6alkyl. In some embodiments, R is methyl. In some embodiments, R is substituted methyl. In some embodiments, R is ethyl. In some embodiments, R is substituted ethyl.

[0212] In some embodiments, as described herein, a non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage.

[0213] In some embodiments, a modified internucleotidic linkage (e.g., a non-negatively charged internucleotidic linkage) comprises optionally substituted triazolyl. In some embodiments, R’ is or comprises optionally substituted triazolyl. In some embodiments, a modified internucleotidic linkage (e.g., a non- negatively charged internucleotidic linkage) comprises optionally substituted alkynyl. In some embodiments, R’ is optionally substituted alkynyl. In some embodiments, R’ comprises an optionally substituted triple bond. In some embodiments, a modified internucleotidic linkage comprises a triazole or alkyne moiety. In some embodiments, R’ is or comprises an optionally substituted triazole or alkyne moiety. In some embodiments, a triazole moiety, e.g., a triazolyl group, is optionally substituted. In some embodiments, a triazole moiety, e.g., a triazolyl group) is substituted. In some embodiments, a triazole moiety is unsubstituted. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted guanidine moiety. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted cyclic guanidine moiety. In some embodiments, R’, RL, or −X−RL, is or comprises an optionally substituted guanidine moiety. In some embodiments, R’, RL, or −X−RL, is or comprises an optionally substituted cyclic guanidine moiety. In some embodiments, R’, RL, or −X−RLcomprises an optionally substituted cyclic guanidine moiety and an Page 101 of 274 12621671v1Attorney Docket No.: 2010581-1497 internucleotidic linkage has the structure , wherein W is O or S. In some embodiments, a non-negatively charged internucleotidic

[0214] In some embodiments, a non-negatively charged internucleotidic linkage or a neutral internucleotidic linkage is an internucleotidic linkage comprising a triazole moiety. In some embodiments, a non-negatively charged internucleotidic linkage or a non-negatively charged internucleotidic linkage comprises an optionally substituted triazolyl group. In some embodiments, an internucleotidic linkage comprising a triazole moiety (e.g., an optionally substituted triazolyl group) has the . In someembodiments, an internucleotidic linkage comprising a triazole moiety has the structure . In some embodiments, an internucleotidic linkage, e.g., a non-negatively chargeda neutral internucleotidic linkage, comprises a cyclic guanidine moiety. In some embodiments, an internucleotidic linkage comprising a cyclic guanidine moiety has the In some embodiments, a non-negatively charged internucleotidic linkage, or ais or comprising a structure selected from , or, wherein W is O or S.

[0215] In some embodiments, an internucleotidic linkage comprises a Tmg ). In somePage 102 of 274 12621671v1Attorney Docket No.: 2010581-1497embodiments, an internucleotidic linkage comprises a Tmg group and has(the “Tmg internucleotidic linkage”). In some embodiments, neutral internucleotidic linkages of PNA and PMO, and an Tmg internucleotidic

[0216] In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, such a heterocyclyl or heteroaryl group is of a 5-membered ring. In some embodiments, such a heterocyclyl or heteroaryl group is of a 6-membered ring.

[0217] In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-6 membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a heteroaryl group is directly bonded to a linkage phosphorus. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heterocyclyl group having 1-10 heteroatoms. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heterocyclyl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-6 membered heterocyclyl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5- membered heterocyclyl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, at least two heteroatoms are nitrogen. In some embodiments, a heterocyclyl group is directly bonded to a linkage phosphorus. In some embodiments, a heterocyclyl group is bonded to a linkage phosphorus through a linker, e.g., =N− when the heterocyclyl group is part of a guanidine moiety who directed bonded to a linkage phosphorus through its =N−. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted group. In some embodiments, a non-negatively charged internucleotidic linkage comprises an substituted group. In some embodiments, a non-negatively Page 103 of 274 12621671v1Attorney Docket No.: 2010581-1497 charged internucleotidic linkage group. In some embodiments, each R1is independently optionally substituted C1-6 each R1is independently methyl.

[0218] In some embodiments, ainternucleotidic linkage, e.g., a neutral internucleotidic linkage is not chirally controlled. In some embodiments, a non-negatively charged internucleotidic linkage is chirally controlled. In some embodiments, a non-negatively charged internucleotidic linkage is chirally controlled and its linkage phosphorus is Rp. In some embodiments, a non-negatively charged internucleotidic linkage is chirally controlled and its linkage phosphorus is Sp.

[0219] In some embodiments, an internucleotidic linkage comprises no linkage phosphorus. In some embodiments, an internucleotidic linkage has the structure of −C(O)−(O)− or −C(O)−N(R’)−, wherein R’ is as described herein. In some embodiments, an internucleotidic linkage has the structure of −C(O)−(O)−. In some embodiments, an internucleotidic linkage has the structure of −C(O...

Claims

Attorney Docket No.: 2010581-1497 CLAIMS 1. An oligonucleotide, comprising: 5’−L-1−N1−L1−N2−L2−N3−L3−3’, wherein: each of N1, N2 and N3 is independently a nucleoside; the sugar of each of N1, N2and N3is independently a sugar comprising no 2’-modification; L-1is a Rp PN internucleotidic linkage; L1is a Rp PS internucleotidic linkage or a PO internucleotidic linkage; and each of L2and L3is independently a Sp PS internucleotidic linkage.

2. An oligonucleotide, comprising: a PN internucleotidic linkage bonded to a sugar comprising no 2’-modification; and a PS internucleotidic linkage bonded to a sugar comprising no 2’-modification.

3. An oligonucleotide, comprising: (I) 5’−N-1−L-1−N1−L1−N2−L2−N3−L3−3’, wherein: each of N-1, N1, N2 and N3 is independently a nucleoside; the sugar of each of N-1, N1, N2 and N3 is independently a sugar comprising no 2’-modification; L-1is a Rp PN internucleotidic linkage; L1is a PO or Rp PS internucleotidic linkage; and each of L2and L3is independently a Sp PS internucleotidic linkage; or (II) 5’−L-2−N-1−L-1−N1−L1−N2−L2−N3−L3−3’, wherein: each of N-1, N1, N2 and N3 is independently a nucleoside; the sugar of each of N-1, N1, N2 and N3 is independently a sugar comprising no 2’-modification; each of L-2and L-1is independently a Rp PN or Sp PS internucleotidic linkage; at least one of L-2and L-1is a Rp PN internucleotidic linkage; L1is a PO or Rp PS internucleotidic linkage; and each of L2and L3is independently a Sp PS internucleotidic linkage; or (III) 5’−N-2−L-2−N-1−L-1−N1−L1−N2−L2−N3−L3−3’, wherein: each of N-2, N-1, N1, N2and N3is independently a nucleoside; the sugar of each of N-2, N-1, N1, N2and N3is independently a sugar comprising no 2’-modification. each of L-2and L-1is independently a Rp PN or Sp PS internucleotidic linkage; at least one of L-2and L-1is a Rp PN internucleotidic linkage; L1is a PO or Rp PS internucleotidic linkage; and each of L2and L3is independently a Sp PS internucleotidic linkage; or Page 265 of 274 12621671v1Attorney Docket No.: 2010581-1497 (III) 5’−L-8−N-7−L-7−N-6−L-6−N-5−L-5−N-4−L-4−N-3−L-3−N-2−L-2−N-1−L-1−N1−L1−N2−L2−N3−L3−3’, wherein: each of N-7, N-6, N-5, N-4, N-3, N-2, N-1, N1, and N2is independently a nucleoside; and each of L-8, L-7, L-6, L-5, L-4, L-3, L-2, L-1, L1, L2, and L3is independently an internucleotidic linkage; or (IV) 5’−N-8−L-8−N-7−L-7−N-6−L-6−N-5−L-5−N-4−L-4−N-3−L-3−N-2−L-2−N-1−L-1−N1−L1−N2−L2−N3−L3−N4−3’, wherein: each of N-8, N-7, N-6, N-5, N-4, N-3, N-2, N-1, N1, N2, and N3 is independently a nucleoside; and each of L-8, L-7, L-6, L-5, L-4, L-3, L-2, L-1, L1, L2, and L3is independently an internucleotidic linkage.

4. The oligonucleotide of claim 3, wherein N-8 is the 5thnucleoside from the 5’-end of the oligonucleotide.

5. The oligonucleotide of any one of the preceding claims, wherein the sugar of each of N1, N2 and N3 is independently a natural DNA sugar.

6. The oligonucleotide of any one of claims 1-5, wherein the sugar of N-1 is a natural DNA sugar.

7. The oligonucleotide of any one of claims 3-6, wherein the sugar of N-2 is a natural DNA sugar.

8. The oligonucleotide of any one of claims claim 3-7, wherein the sugar of N-3 is a natural DNA sugar.

9. The oligonucleotide of any one of claims 3-8, wherein the sugar of N-4 is a natural DNA sugar.

10. The oligonucleotide of any one of claims 3-9, wherein the sugar of N-5 is a natural DNA sugar.

11. The oligonucleotide of any one of claims claim 3-10, wherein the sugar of N-6 is a natural DNA sugar.

12. The oligonucleotide of any one of claims 3-11, wherein the sugar of N-7 is a natural DNA sugar.

13. The oligonucleotide of any one of claims 3-12, wherein the sugar of N-8 is a 2’-modified sugar.

14. The oligonucleotide of claim 13, wherein the sugar of N-8 is a 2’-ORsamodified sugar, wherein Rsais C1-6 aliphatic.

15. The oligonucleotide of any one of the preceding claims, wherein L1is a Rp PS internucleotidic linkage.

16. The oligonucleotide of any one of claims 3-14, wherein L1is a PO internucleotidic linkage.

17. The oligonucleotide of any one of the preceding claims, wherein L2is a Sp PS internucleotidic linkage.

18. The oligonucleotide of any one of the preceding claims, wherein L3is a Sp PS internucleotidic linkage.

19. The oligonucleotide of any one of the preceding claims, wherein L-1is a Sp PS internucleotidic linkage.

20. The oligonucleotide of any one of claims 1-18, wherein L-1is a Rp PN internucleotidic linkage.

21. The oligonucleotide of any one of claims 1-18, wherein L-1is a PN internucleotidic linkage.

22. The oligonucleotide of any one of the preceding claims, wherein L-2is a Sp PS internucleotidic linkage. Page 266 of 274 12621671v1Attorney Docket No.: 2010581-1497 23. The oligonucleotide of any one of claims 3-21, wherein L-2is a Rp PN internucleotidic linkage.

24. The oligonucleotide of any one of claims 3-21, wherein L-2is a PN internucleotidic linkage.

25. The oligonucleotide of any one of claims 3-24, wherein L-3is a Rp PS internucleotidic linkage, a Sp PS internucleotidic linkage, a Rp PN internucleotidic linkage, or a Sp PN internucleotidic linkage.

26. The oligonucleotide of any one of claims 3-25, wherein L-4is a Rp PS internucleotidic linkage, a Sp PS internucleotidic linkage, a Rp PN internucleotidic linkage, or a Sp PN internucleotidic linkage.

27. The oligonucleotide of any one of claims 3-26, wherein L-5is a Rp PS internucleotidic linkage, a Sp PS internucleotidic linkage, a Rp PN internucleotidic linkage, or a Sp PN internucleotidic linkage.

28. The oligonucleotide of any one of claims 3-27, wherein L-6is a Rp PS internucleotidic linkage , a Sp PS internucleotidic linkage, a Rp PN internucleotidic linkage, or a Sp PN internucleotidic linkage.

29. The oligonucleotide of any one of claims 3-28, wherein L-7is a Rp PS internucleotidic linkage, a Sp PS internucleotidic linkage, a Rp PN internucleotidic linkage, or a Sp PN internucleotidic linkage.

30. The oligonucleotide of any one of claims 3-29, wherein L-8is a Rp PS internucleotidic linkage, a Sp PS internucleotidic linkage, a Rp PN internucleotidic linkage, or a Sp PN internucleotidic linkage.

31. The oligonucleotide of any one of the preceding claims, comprising 5’-wing-core-wing-3’.

32. The oligonucleotide of any one of claims 3-31, wherein N-7 is the first nucleoside in a core from the 5’-end and / or N3 is the last nucleoside in a core from the 5’-end.

33. An oligonucleotide, wherein the oligonucleotide comprises 5’-wing-core-wing-3’, wherein the oligonucleotide comprises a Rp or Sp PN internucleotidic linkage bonded to a sugar comprising no 2’- modification.

34. The oligonucleotide of any one of the preceding claims, wherein the 5’-wing has a length of 2 or more nucleobases.

35. The oligonucleotide of any one of the preceding claims, wherein the 5’-wing comprises one or more modified sugars.

36. The oligonucleotide of claim 35, wherein each sugar in the 5’-wing is independently a modified sugar.

37. The oligonucleotide of any one of claims 35-36, wherein a modified sugar in the 5’-wing comprises a 2’-modification.

38. The oligonucleotide of any one of claims 35-36, wherein a modified sugar in the 5’-wing is a 2’-OMe or 2’-MOE modified sugar.

39. The oligonucleotide of any one of the preceding claims, wherein the 5’-wing comprises a PN internucleotidic linkage.

40. The oligonucleotide of any one of the preceding claims, wherein the 5’-wing comprises a PS internucleotidic linkage.

41. The oligonucleotide of any one of claims 31-40, wherein the core has a length of 5 or more nucleobases. Page 267 of 274 12621671v1Attorney Docket No.: 2010581-1497 42. The oligonucleotide of any one of claims 31-41, wherein the core has a length of 10 nucleobases.

43. The oligonucleotide of any one of claims 31-42, wherein each sugar of the core comprises no 2’- modification.

44. The oligonucleotide of any one of the preceding claims, wherein each sugar comprising no 2’- modification is a natural DNA sugar.

45. The oligonucleotide of any one of claims 31-42, wherein a sugar of the core is a modified sugar.

46. The oligonucleotide of claim 45, wherein the number of modified sugars in the core is about or no more than about 3.

47. The oligonucleotide of any one of claims 31-46, wherein the core comprises no phosphate internucleotidic linkage.

48. The oligonucleotide of any one of claims 31-47, wherein the core comprises a PN internucleotidic linkage.

49. The oligonucleotide of any one of claims 31-47, wherein the core comprises two, three, four or five consecutive PN internucleotidic linkages.

50. The oligonucleotide of any one of claims 31-47, wherein the core comprises a PN internucleotidic linkage and a PS internucleotidic linkage.

51. The oligonucleotide of any one of claims 31-50, wherein the pattern of internucleotidic linkages of the oligonucleotide comprises: (1) (NR)tSRSSSS, wherein t is 1, 2, 3, 4, or 5, SR represents a PS linkage in Rp configuration, each SS independently represents a PS linkage in Sp configuration, each NR independently represents a PN linkage in Rp configuration, and each internucleotidic linkage of the (NR)tSRSSSS independently bonds to a sugar in the core, (2) SRSSSS, wherein SR represents a PS linkage in Rp configuration, each SS independently represents a PS linkage in Sp configuration, and each internucleotidic linkage of the SRSSSS independently bonds to a sugar in the core, (3) NRSRSSSS, wherein SR represents a PS linkage in Rp configuration, each SS independently represents a PS linkage in Sp configuration, NR represents a PN linkage in Rp configuration, and each internucleotidic linkage of the NRSRSSSSindependently bonds to a sugar in the core, (4) NRNRSRSSSS, wherein SRrepresents a PS linkage in Rp configuration, each SSindependently represents a PS linkage in Sp configuration, NRrepresents a PN linkage in Rp configuration, and each internucleotidic linkage of the NRNRSRSSSSindependently bonds to a sugar in the core, (5) SRNSSS, wherein SRrepresents a PS linkage in Rp configuration, SSrepresents a PS linkage in Sp configuration, NSrepresents a PN linkage in Sp configuration, and each internucleotidic linkage of the SRNSSSindependently bonds to a sugar in the core, (6) (NR)2SRSSSS, wherein SRrepresents a PS linkage in Rp configuration, each SSindependently represents a PS linkage in Sp configuration, each NRindependently represents a PN linkage in Rp Page 268 of 274 12621671v1Attorney Docket No.: 2010581-1497 configuration, and each internucleotidic linkage of the (NR)2SRSSSSindependently bonds to a sugar in the core, or (7) (NR)tSR(SS)n, wherein t is 1, 2, 3, 4, or 5, n is 2, 3, 4, 5, 6, 7 or 8, SRrepresents a PS linkage in Rp configuration, each SSindependently represents a PS linkage in Sp configuration, each NRindependently represents a PN linkage in Rp configuration, and each internucleotidic linkage of the (NR)tSR(SS)n independently bonds to a sugar in the core.

52. The oligonucleotide of any one of claims 31-51, wherein the pattern of internucleotidic linkages of the oligonucleotide comprises: (1) (Ln)m(Ln)tSR(SS)n, wherein m is 1, 2, 3, 4 or 5, t is 1, 2, 3, 4, or 5, n is 2, 3, 4, 5, 6, 7 or 8, each Lnis independently SR, SS, NR or NS, each SR independently represents a PS linkage in Rp configuration, each SS independently represents a PS linkage in Sp configuration, each NR independently represents a PN linkage in Rp configuration, each NS independently represents a PN linkage in Sp configuration, and each internucleotidic linkage of the (Ln)m(Ln)tSR(SS)n independently bonds to a sugar in the core, or (2) (Ln)m(NR)tSR(SS)n, wherein m is 1, 2, 3, 4 or 5, t is 1, 2, 3, 4, or 5, n is 2, 3, 4, 5, 6, 7 or 8, each Lnis independently SR, SS, NR or NS, each SR independently represents a PS linkage in Rp configuration, each SS independently represents a PS linkage in Sp configuration, each NR independently represents a PN linkage in Rp configuration, each NS independently represents a PN linkage in Sp configuration, and each internucleotidic linkage of the (Ln)m(NR)tSR(SS)n independently bonds to a sugar in the core.

53. The oligonucleotide of claim 51, wherein each Lnis independently: (1) NR, NS or SS, (2) NS or SS, (3) NS, or (4) SS.

54. The oligonucleotide of any one of claims 31-53, wherein the pattern of internucleotidic linkages of the oligonucleotide comprises (NS)m(NR)tSR(SS)n, wherein m is 1, 2, 3, 4 or 5, t is 1, 2, 3, 4, or 5, n is 2, 3, 4, 5, 6, 7 or 8, SR represents a PS linkage in Rp configuration, each SS independently represents a PS linkage in Sp configuration, each NR independently represents a PN linkage in Rp configuration, each NS independently represents a PN linkage in Sp configuration, and each internucleotidic linkage of the (NS)m(NR)tSR(SS)nindependently bonds to a sugar in the core.

55. The oligonucleotide of claim 52, wherein (Ln)m(Ln)tSR(SS)nis or comprises: (1) (NS)mNRSRSSSS, (2) (NS)mNRNRSRSSSS, (3) NRNRSRSSSS, (4) NSNRNRSRSSSS, (5) NSNSNRNRSRSSSS, (6) NSNSNSNRNRSRSSSS, Page 269 of 274 12621671v1Attorney Docket No.: 2010581-1497 (7) NSSRSSSS, (8) SSNSSRSSSS, (9) NSSRSSSSSRSSSS, (10) NRSRSSSS, or (11) SSNRSRSSSS.

56. The oligonucleotide of any one of the preceding claims, wherein the SRbetween (Ln)m(Ln)tand (SS)nis L1.

57. The oligonucleotide of any one of the preceding claims, wherein the pattern of internucleotidic linkages of the oligonucleotide comprises: (1) SSSRSSSS, wherein SR represents a PS linkage in Rp configuration, each SS independently represents a PS linkage in Sp configuration, and each internucleotidic linkage of the SSSRSSSS independently bonds to a sugar in the core; or (2) NSSRSSSS, wherein SR represents a PS linkage in Rp configuration, each SS independently represents a PS linkage in Sp configuration, NS represents PN in Sp configuration, and each internucleotidic linkage of the NSSRSSSS independently bonds to a sugar in the core; or (3) (NS)2SRSSSS, wherein SR represents a PS linkage in Rp configuration, each SS independently represents a PS linkage in Sp configuration, each NS independently represents PN in Sp configuration, and each internucleotidic linkage of the (NS)2SRSSSS independently bonds to a sugar in the core; or (4) SRSSSSSRSSSS, wherein each R independently represents a PS linkage in Rp configuration, each SS independently represents a PS linkage in Sp configuration, and each internucleotidic linkage of the SRSSSSSRSSSS independently bonds to a sugar in the core; or (6) SRSSSSSRSSSSSRSSSS, wherein each R independently represents a PS linkage in Rp configuration, each SS independently represents a PS linkage in Sp configuration, and each internucleotidic linkage of the SRSSSSSRSSSSSRSSSS independently bonds to a sugar in the core; or (7) repeating SRSSSS, wherein each R independently represents a PS linkage in Rp configuration, each SS independently represents a PS linkage in Sp configuration, and each internucleotidic linkage of the repeating SRSSSS independently bonds to a sugar in the core.

58. The oligonucleotide of any one of claims 31-57, wherein the core is of the structure 5’−L-8−N-7−L-7−N-6−L-6−N-5−L-5−N-4−L-4−N-3−L-3−N-2−L-2−N-1−L-1−N1−L1−N2−L2−N3−L3−3’.

59. The oligonucleotide of any one of claims 3-58, wherein L-2is a Rp PN internucleotidic linkage, a Sp PN internucleotidic linkage, a Rp PS internucleotidic linkage or a Sp PS internucleotidic linkage.

60. The oligonucleotide of any one of claims 3-59, wherein L-1is a Rp PN internucleotidic linkage, a Sp PN internucleotidic linkage, a Sp PS internucleotidic linkage, or a Rp PS internucleotidic linkage.

61. The oligonucleotide of any one of claims 1-60, wherein L1is a Rp PS internucleotidic linkage.

62. The oligonucleotide of any one of claims 1-60, wherein L1is a PO internucleotidic linkage. Page 270 of 274 12621671v1Attorney Docket No.: 2010581-1497 63. The oligonucleotide of any one of claims 1-62, wherein L2is a Sp PS internucleotidic linkage.

64. The oligonucleotide of any one of claims 3-63, wherein L3is a Sp PS internucleotidic linkage.

65. The oligonucleotide of any one of the preceding claims, wherein L1is bonded to the 8thsugar and the 9thsugar in the core.

66. The oligonucleotide of any one of the preceding claims, wherein the 3’-wing has a length of 2 or more nucleobases.

67. The oligonucleotide of any one of claims 1-66, wherein the 3’-wing comprises one or more modified sugars.

68. The oligonucleotide of any one of claims 1-67 wherein each sugar in the 3’-wing is independently a modified sugar.

69. The oligonucleotide of any one of claims 66-67, wherein a modified sugar in the 3’-wing comprises a 2’-modification.

70. The oligonucleotide of any one of claims 67-69, wherein a modified sugar in the 3’-wing is a 2’-OMe or 2’-MOE modified sugar.

71. The oligonucleotide of any one of the preceding claims, wherein the 3’-wing comprises a PN internucleotidic linkage.

72. The oligonucleotide of any one of the preceding claims, wherein the 3’-wing comprises a Sp PS internucleotidic linkage.

73. The oligonucleotide of any one of the preceding claims, wherein each sugar comprising no 2’- modification in the core is independently a natural DNA sugar.

74. The oligonucleotide of any one of the preceding claims, wherein each PO linkage is independently a natural phosphate linkage.

75. The oligonucleotide of any one of the preceding claims, wherein each PS linkage is independently a phosphorothioate linkage.

76. The oligonucleotide of any one of the preceding claims, wherein a PN linkage is a phosphoramidate linkage.

77. The oligonucleotide of any one of the preceding claims, wherein a PN linkage is a phosphoryl guanidine linkage.

78. The oligonucleotide of any one of the preceding claims, wherein a PN linkage is a n001 linkage.

79. The oligonucleotide of any one of the preceding claims, wherein a PN linkage is a n006 linkage.

80. The oligonucleotide of any one of the preceding claims, wherein a PN linkage is a MsPA linkage.

81. The oligonucleotide of any one of the claims 1-75, wherein each PN linkage is independently a phosphoramidate linkage.

82. The oligonucleotide of any one of the claims 1-75, wherein each PN linkage is independently a phosphoryl guanidine linkage. Page 271 of 274 12621671v1Attorney Docket No.: 2010581-1497 83. The oligonucleotide of any one of the claims 1-80, wherein each PN linkage is independently a n001 linkage, a n006 linkage or a MsPA linkage.

84. The oligonucleotide of any one of the claims 1-80, wherein each PN linkage is independently a n001 linkage.

85. The oligonucleotide of any one of the preceding claims, wherein each chiral internucleotidic linkage is independently a PS or PN internucleotidic linkage.

86. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide is in a salt form.

87. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide is in a pharmaceutically acceptable salt form.

88. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide is in a sodium salt form.

89. A composition, wherein the composition is a pharmaceutical composition comprising an oligonucleotide of any one of the preceding claims and a pharmaceutically acceptable carrier.

90. The composition of claim 89, wherein the composition is chirally controlled.

91. The composition of any one of the preceding claims, wherein about or at least about 5%-100%, 10%- 100%, 20-100%, 30%-100%, 40%-100%, 50%-100%, 5%-90%, 10%-90%, 20-90%, 30%-90%, 40%-90%, 50%-90%, 5%-85%, 10%-85%, 20-85%, 30%-85%, 40%-85%, 50%-85%, 5%-80%, 10%-80%, 20-80%, 30%-80%, 40%-80%, 50%-80%, 5%-75%, 10%-75%, 20-75%, 30%-75%, 40%-75%, 50%-75%, 5%-70%, 10%-70%, 20-70%, 30%-70%, 40%-70%, 50%-70%, 5%-65%, 10%-65%, 20-65%, 30%-65%, 40%-65%, 50%-65%, 5%-60%, 10%-60%, 20-60%, 30%-60%, 40%-60%, 50%-60%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the composition that share the constitution of the oligonucleotide are the oligonucleotide.

92. The composition of any one of the preceding claims, wherein the stereochemical purity of each PS internucleotidic linkage of the oligonucleotide is independently about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% or more; and / or wherein the stereochemical purity of each PN internucleotidic linkage of the oligonucleotide is independently about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% or more; and / or wherein the stereochemical purity of each chiral linkage phosphorus of the oligonucleotide is independently about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% or more.

93. A method for reducing level of a RNA or a product thereof in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide or composition of any one of the preceding claims, wherein the base sequence of the oligonucleotide is complementary to that of the RNA or a portion thereof; or a method for reducing level of a RNA or a product thereof in a system, comprising administering or delivering to the system an effective amount of an oligonucleotide or composition of any one of the preceding claims, wherein the oligonucleotide can hybridize to the RNA or a portion thereof. Page 272 of 274 12621671v1Attorney Docket No.: 2010581-1497 94. A method for preventing or treating a condition, disorder or disease, comprising administering or delivering to a subject suffering therefrom an oligonucleotide or composition of any one of the preceding claims, wherein the condition, disorder or disease is associated with level of a RNA or a polypeptide encoded thereby, wherein the base sequence of the oligonucleotide is complementary to that of the RNA or a portion thereof; or a method for preventing or treating a condition, disorder or disease, comprising administering or delivering to a subject suffering therefrom an effective amount of an oligonucleotide or composition of any one of the preceding claims, wherein the condition, disorder or disease is associated with level of a RNA or a polypeptide encoded thereby, wherein the oligonucleotide can hybridize to the RNA or a portion thereof.

95. The method of any one of the preceding claims, wherein the RNA is a transcript of TARDBP.

96. A method for preparing an oligonucleotide or composition of any one of the preceding claims, comprising utilizing a phosphoramidite (e.g., a phosphoramidite comprising a chiral auxiliary as described herein).

97. An oligonucleotide or composition of any one of the preceding claims, for a method of any one of the preceding claims, or for manufacturing a medicament for a method of any one of the preceding claims.

98. Use of an oligonucleotide or composition of any one of the preceding claims, for a method of any one of the preceding claims, or for manufacturing a medicament for a method of any one of the preceding claims.

99. An oligonucleotide, composition, method or use of any one of Embodiments 1-428. Page 273 of 274 12621671v1

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