OLIGONUCLEOTIDE COMPOSITIONS AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application Nos.62/911,334, filed October 6, 2019, 62/959,917, filed January 11, 2020, 63/022,559, filed May 10, 2020, and 63/069,696, filed August 24, 2020, the entirety of each of which is incorporated herein by reference.

BACKGROUND

[0002] 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

[0003] Among other things, the present disclosure provides designed oligonucleotides and compositions thereof which oligonucleotides comprise modifications (e.g., modifications to nucleobases sugars, and/or internucleotidic linkages, and patterns thereof) as described herein. In some embodiments, technologies (compounds (e.g., oligonucleotides), compositions, methods, etc.) of the present disclosure (e.g., oligonucleotides, oligonucleotide compositions, methods, etc.) are particularly useful for editing nucleic acids, e.g., site-directed editing in nucleic acids (e.g., editing of target adenosine). In some embodiments, as demonstrated herein, provided technologies can significantly improve efficiency of nucleic acid editing, e.g., modification of one or more A residues, such as conversion of A to I. In some embodiments, the present disclosure provides technologies for editing (e.g., for modifying an A residue, e.g., converting an A to I) in an RNA. In some embodiments, the present disclosure provides technologies for editing (e.g., for modifying an A residue, e.g., converting an A to an I) in a transcript, e.g., mRNA. Among other things, provided technologies provide the benefits of utilization of endogenous proteins such as ADAR (Adenosine Deaminases Acting on RNA) proteins (e.g., ADAR1 and/or ADR2), for editing nucleic acids, e.g., for modifying an A (e.g., as a result of G to A mutation). Those skilled in the art will appreciates that such utilization of endogenous proteins can avoid a number of challenges and/or provide various benefits compared to those technologies that require the delivery of exogenous components (e.g., proteins (e.g., those engineered to bind to oligonucleotides (and/or duplexes thereof with target nucleic acids) to provide desired activities), nucleic acids encoding proteins, viruses, etc.).

[0004] Particularly, in some embodiments, oligonucleotides of provided technologies comprise useful sugar modifications and/or patterns thereof (e.g., presence and/or absence of certain modifications), nucleobase modifications and/or patterns thereof (e.g., presence and/or absence of certain modifications), internucleotidic linkages modifications and/or stereochemistry and/or patterns thereof [e.g., types, modifications, and/or configuration (Rp or Sp) of chiral linkage phosphorus, etc.], etc., which, when combined with one or more other structural elements described herein (e.g., additional chemical moieties) can provide high activities and/or various desired properties, e.g., high efficiency of nucleic acid editing, high selectivity, high stability, high cellular uptake, low immune stimulation, low toxicity, improved distribution, improved affinity, etc. In some embodiments, provided oligonucleotides provide high stability, e.g., when compared to oligonucleotides having a high percentage of natural RNA sugars utilized for adenosine editing. In some embodiments, provided oligonucleotides provide high activities, e.g., adenosine editing activity. In some embodiments, provided oligonucleotides provide high selectivity, for example, in some embodiments, provided oligonucleotides provide selective modification of a target adenosine in a target nucleic acid over other adenosine in the same target nucleic acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 fold or more modification at the target adenosine than another adenosine, or all other adenosine, in a target nucleic acid).

[0005] In some embodiments, the present disclosure provides an oligonucleotide comprising a first domain and a second domain, wherein the first domain comprises one or more 2’-F modifications, and the second domain comprises one or more sugars that do not have a 2’-F modification. In some embodiments, a provided oligonucleotide comprises one or more chiral modified internucleotidic linkages. In some embodiments, the present disclosure provides an oligonucleotide comprising:

(a) a first domain; and

(b) a second domain,

wherein the first domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more sugars comprising a 2’-F modification, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all sugars of the first domain comprises a 2’-F modification;

the second domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more modified sugars comprising no 2’-F modification, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all sugars of the second domain comprise no 2’-F modification.

[0006] In some embodiments, a second domain comprises or consists of a first subdomain, a second subdomain and a third subdomain as described herein.

[0007] In some embodiments, a second domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more modified sugars independently comprising a 2’-OR modification, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all sugars of a second domain comprise a 2’-OR modification, wherein R is optionally substituted C1-6 aliphatic. In some embodiments, R is methyl. In some embodiments, R is CH2CH2OCH3. As described herein, other sugar modifications may also be utilized in accordance with the present disclosure, optionally with base modifications and/or

internucleotidic linkage modifications described herein.

[0008] In some embodiments, base sequence of a provided oligonucleotide is substantially complementary to the base sequence of a target nucleic acid comprising a target adenosine. In some embodiments, a provided oligonucleotide when aligned to a target nucleic acid comprises one or more mismatches (non-Watson-Crick base pairs). In some embodiments, a provided oligonucleotide when aligned to a target nucleic acid comprises one or more wobbles (e.g., G-U, I-A, G-A, I-U, I-C, etc.). In some embodiments, mismatches and/or wobbles may help one or more proteins, e.g., ADAR1, ADAR2, etc., to recognize a duplex formed by a provided oligonucleotide and a target nucleic acid. In some embodiments, provided oligonucleotides form duplexes with target nucleic acids. In some embodiments, ADAR proteins recognize and bind to such duplexes. In some embodiments, nucleosides opposite to target adenosines are located in the middle of provided oligonucleotides, e.g., with 5-50 nucleosides to 5’ side, and 1-50 nucleosides on its 3’ side. In some embodiments, a 5’ side has more nucleosides than a 3’ side. In some embodiments, a 5’ side has fewer nucleosides than a 3’ side. In some embodiments, a 5’ side has the same number of nucleosides as a 3’ side. In some embodiments, provided oligonucleotides comprise 15-40, e.g., 15, 20, 25, 30, etc. contiguous bases of oligonucleotides described in the Tables. In some embodiments, base sequences of provided oligonucleotides are or comprises base sequences of oligonucleotides described in the Tables.

[0009] In some embodiments, with utilization of various structural elements (e.g., various modifications, stereochemistry, and patterns thereof), the present disclosure can achieve desired properties and high activities with short oligonucleotides, e.g., those of about 20-40, 25-40, 25-35, 26-32, 25, 26, 27, 28, 29, 30, 31, 3233, 34 or 35 nucleobases in length.

[0010] In some embodiments, provided oligonucleotides comprise modified nucleobases. In some embodiments, a modified nucleobase promotes modification of a target adenosine. In some embodiments, a nucleobase which is opposite to a target adenine maintains interactions with an enzyme, e.g., ADAR, compared to when a U is present, while interacts with a target adenine less strongly than U (e.g., forming fewer hydrogen bonds). In some embodiments, an opposite nucleobase and/or its associated sugar provide certain flexibility (e.g., when compared to U) to facility modification of a target adenosine by enzymes, e.g., ADAR1, ADAR2, etc. In some embodiments, a nucleobase immediately 5’ or 3’ to the opposite nucleobase (to a target adenine), e.g., I and derivatives thereof, enhances modification of a target adenine. Among other things, the present disclosure recognizes that such a nucleobase may causes less steric hindrance than G when a duplex of a provided oligonucleotide and its target nucleic acid interact with a modifying enzyme, e.g., ADAR1 or ADAR2. In some embodiments, base sequences of oligonucleotides are selected (e.g., when several adenosine residues are suitable targets) and/or designed (e.g., through utilization of various nucleobases described herein) so that steric hindrance may be reduced or removed (e.g., no G next to the opposite nucleoside of a target A).

[0011] In some embodiments, oligonucleotides of the present disclosure provides modified internucleotidic linkages (i.e., internucleotidic linkages that are not natural phosphate linkages). In some embodiments, linkage phosphorus of modified internucleotidic linkages (e.g., chiral internucleotidic linkages) are chiral and can exist in different configurations (Rp and Sp). Among other things, the present disclosure demonstrates that incorporation of modified internucleotidic linkage, particularly with control of stereochemistry of linkage phosphorus centers (so that at such a controlled center one configuration is enriched compared to stereorandom oligonucleotide preparation), can significantly improve properties (e.g., stability) and/or activities (e.g., adenosine modifying activities (e.g., converting an adenosine to inosine). In some embodiments, provided oligonucleotides have stereochemical purity significantly higher than stereorandom preparations. In some embodiments, provided oligonucleotides are chirally controlled.

[0012] In some embodiments, oligonucleotides of the present disclosure comprise one or more chiral internucleotidic linkages whose linkage phosphorus is chiral (e.g., a phosphorothioate internucleotidic linkage). In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all internucleotidic linkages in an oligonucleotide, are chiral internucleotidic linkages. In some embodiments, at least one internucleotidic linkage is a chiral internucleotidic linkage. In some embodiments, at least one internucleotidic linkage is a natural phosphate linkage. In some embodiments, each internucleotidic linkage is independently a chiral internucleotidic linkage. In some embodiments, at least one chiral internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, each is a phosphorothioate internucleotidic linkage. A linkage phosphorus can be either Rp or Sp. In some embodiments, at least one linkage phosphorus is Rp. In some embodiments, at least one linkage phosphorus is Sp. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all chiral internucleotidic linkages in an oligonucleotide, are Sp. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all phosphorothioate internucleotidic linkages in an oligonucleotide, are Sp.

[0013] In some embodiments, stereochemistry of one or more chiral linkage phosphorus of provided oligonucleotides are controlled in a composition. In some embodiments, the present disclosure provides a composition comprising a plurality of oligonucleotides, wherein oligonucleotides of a plurality share a common base sequence, and the same configuration of linkage phosphorus (e.g., all are Rp or all are Sp for the chiral linkage phosphorus) independently at one or more (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30, 5-25, 5-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all

chiral internucleotidic linkages) chiral internucleotidic linkages (“chirally controlled internucleotidic linkages”). In some embodiments, they share the same stereochemistry at each chiral linkage phosphorus. In some embodiments, oligonucleotides of a plurality share the same constitution. In some embodiments, oligonucleotides of a plurality are structurally identical except the internucleotidic linkages. In some embodiments, oligonucleotides of a plurality are structurally identical. In some embodiments, at least at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides in a composition, or of all oligonucleotides sharing the common base sequence, share the pattern of backbone chiral centers of oligonucleotides of the plurality. In some embodiments, at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides in a composition, or of all oligonucleotides sharing the common base sequence, are oligonucleotides of the plurality.

[0014] In some embodiments, the present disclosure provides a chirally controlled oligonucleotide composition of an oligonucleotide, wherein at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides in a composition, or of all oligonucleotides having the same base sequence of the oligonucleotide, or of all oligonucleotide having the same base sequence and sugar and base modifications, or of all oligonucleotides of the same constitution, share the same configuration of linkage phosphorus (e.g., all are Rp or all are Sp for the chiral linkage phosphorus) independently at one or more (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30, 5-25, 5-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all chiral internucleotidic linkages) chiral internucleotidic linkages with the oligonucleotide. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide composition of an oligonucleotide, wherein at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides in a composition, or of all oligonucleotides having the same base sequence of the oligonucleotide, or of all oligonucleotide having the same base sequence and sugar and base modifications, or of all oligonucleotides of the same constitution, are one or more forms of the oligonucleotide (e.g., acid forms, salt forms (e.g. pharmaceutically acceptable salt forms; as appreciated by those skilled in the art, in case the oligonucleotide is a salt, other salt forms of the corresponding acid or base form of the oligonucleotide), etc.).

[0015] In some embodiments, as demonstrated herein chirally controlled oligonucleotide compositions provide a number of advantages, e.g., higher stability, activities, etc., compared to corresponding stereorandom oligonucleotide compositions. In some embodiments, it was observed that chirally controlled oligonucleotide compositions provide high levels of adenosine modifying (e.g., converting A to I) activities with various isoforms of an ADAR protein (e.g., p150 and p110 forms of ADAR1) while corresponding stereorandom compositions provide high levels of adenosine modifying (e.g., converting A to I) activities with only certain isoforms of an ADAR protein (e.g., p150 isoform of

ADAR1).

[0016] In some embodiments, provided oligonucleotides comprise an additional moiety, e.g., a targeting moiety, a carbohydrate moiety, etc. In some embodiments, an additional moiety is or comprises a ligand for an asialoglycoprotein receptor. In some embodiments, an additional moiety is or comprises GalNAc or derivatives thereof. Among other things, additional moieties may facilitate delivery to certain target locations, e.g., cells, tissues, organs, etc. (e.g., locations comprising receptors that interact with additional moieties). In some embodiments, additional moieties facilitate delivery to liver.

[0017] In some embodiments, the present disclosure provides technologies for preparing oligonucleotides and compositions thereof, particularly chirally controlled oligonucleotide compositions. In some embodiments, provided oligonucleotides and compositions thereof are of high purity. In some embodiments, oligonucleotides of the present disclosure are at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% stereochemically pure at linkage phosphorus of chiral internucleotidic linkages. In some embodiments, oligonucleotides of the present disclosure are prepared stereoselectively and are substantially free of stereoisomers. In some embodiments, in provided compositions comprising a plurality of oligonucleotides which share the same base sequence of the same pattern of chiral linkage phosphorus stereochemistry (e.g., comprising one or more of Rp and/or Sp, wherein each chiral linkage phosphorus is independently Rp or Sp), at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all oligonucleotides in the composition that share the same base sequence as oligonucleotides of the plurality share the same pattern of chiral linkage phosphorus stereochemistry or are oligonucleotides of the plurality. In some embodiments, in provided compositions comprising a plurality of oligonucleotides which share the same base sequence of the same pattern of chiral linkage phosphorus stereochemistry, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all oligonucleotides in the composition that share the same constitution as oligonucleotides of the plurality share the same pattern of chiral linkage phosphorus stereochemistry or are oligonucleotides of the plurality.

[0018] In some embodiments, the present disclosure describes useful technologies for assessing oligonucleotide and compositions thereof. For example, various technologies of the present disclosure are useful for assessing adenosine modification. As appreciated by those skilled in the art, in some embodiments, modification/editing of adenosine can be assessed through sequencing, mass spectrometry, assessment (e.g., levels, activities, etc.) of products (e.g., RNA, protein, etc.) of modified nucleic acids (e.g., wherein adenosines of target nucleic acids are converted to inosines), etc., optionally in view of other components (e.g., ADAR proteins) presence in modification systems (e.g., an in vitro system, an ex vivo system, cells, tissues, organs, organisms, subjects, etc.). Those skilled in the art will appreciate that oligonucleotides which provide adenosine modification of a target nucleic acid can also provide modified nucleic acid (e.g., wherein a target adenosine is converted into I) and one or more products thereof (e.g.,

mRNA, proteins, etc.). Certain useful technologies are described in the Examples.

[0019] As described herein, oligonucleotides and compositions of the present disclosure may be provided/utilized in various forms. In some embodiments, the present disclosure provides compositions comprising one or more forms of oligonucleotides, e.g., acid forms (e.g., in which natural phosphate linkages exist as –O(P(O)(OH)−O−, phosphorothioate internucleotidic linkages exist as – O(P(O)(SH)−O−), base forms, salt forms (e.g., in which natural phosphate linkages exist as salt forms (e.g., sodium salt (–O(P(O)(O−Na+)−O−), phosphorothioate internucleotidic linkages exist as salt forms (e.g., sodium salt (–O(P(O)(S−Na+)−O−) etc. As appreciated by those skilled in the art, oligonucleotides can exist in various salt forms, including pharmaceutically acceptable salts, and in solutions (e.g., various aqueous buffering system), cations may dissociate from anions. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a provided oligonucleotide and/or one or more pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier. In some embodiments, pharmaceutical compositions are chirally controlled oligonucleotide compositions.

[0020] Provided technologies can be utilized for various purposes. For example, those skilled in the art will appreciate that provided technologies are useful for many purposes involving modification of adenosine, e.g., correction of G to A mutations, modulate levels of certain nucleic acids and/or products encoded thereby (e.g., reducing levels of proteins by introducing A to G/I modifications), modulation of splicing, modulation of translation (e.g., modulating translation start and/or stop site by introducing A to G/I modifications), etc.

[0021] In some embodiments, the present disclosure provides technologies for preventing or treating a condition, disorder or disease that is amenable to an adenosine modification, e.g. conversion of A to I or G. As appreciated by those skilled in the art, I may perform one or more functions of G, e.g., in base pairing, translation, etc. In some embodiments, a G to A mutation may be corrected through conversion of A to I so that one or more products, e.g., proteins, of the G-version nucleic acid can be produced. In some embodiments, the present disclosure provides technologies for preventing or treating a condition, disorder or disease associated with a mutation, comprising administering to a subject susceptible thereto or suffering therefrom a provided oligonucleotide or composition thereof, which oligonucleotide or composition can edit a mutation. In some embodiments, the present disclosure provides technologies for preventing or treating a condition, disorder or disease associated with a G to A mutation, comprising administering to a subject susceptible thereto or suffering therefrom a provided oligonucleotide or composition thereof, which oligonucleotide or composition can modify an A. In some embodiments, provided technologies modify an A in a transcript, e.g., RNA transcript. In some embodiments, an A is converted into an I. In some embodiments, during translation protein synthesis machineries read I as G. In some embodiments, an A form encodes one or more proteins that have one or more higher desired activities and/or one or more better desired properties compared those encoded by its corresponding G form. In some embodiments, an A form provides higher levels, compared to its corresponding G form, of one or more proteins that have one or more higher desired activities and/or one or more better desired properties. In some embodiments, products encoded by an A form are structurally different (e.g., longer, in some embodiments, full length proteins) from those encoded by its corresponding G form. In some embodiments, an A form provides structurally identical products (e.g., proteins) compared to its corresponding G form.

[0022] As those skilled in the art will appreciate, many conditions, disorders or diseases are associated with mutations that can be modified by provided technologies and can be prevented and/or treated using provided technologies. For example, it is reported that there are over 20,000 conditions, disorders or diseases are associated with G to A mutation and can benefit from A to I editing.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1. Provided technologies with various sugar modification patterns can provide desired activities. (a) ADAR1- and (b) ADAR2-mediated editing. Oligonucleotides all have the same sequence targeting a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 and ADAR2, respectively, luciferase reporter construct and indicated compositions. cLuc activity was measure and normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0024] Figure 2. Provided technologies comprising various internucleotidic linkage modifications can provide desired activities. (a) and (b): Compositions all have the same sequence targeting a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 or ADAR2, luciferase reporter construct and indicated compositions. cLuc activity was measure and normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0025] Figure 3. Provided technologies comprising various sugar modifications can provide desired activities. Compositions all target a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 or ADAR2, luciferase reporter construct and indicated compositions. cLuc activity was measure and normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0026] Figure 4. Provided technologies comprising various sugar types can provide desired activities. Compositions all have the same sequence targeting a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 or ADAR2, luciferase reporter construct and indicated compositions. cLuc activity was measure and normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0027] Figure 5. Provided technologies can provide desired activities with short sequences. Compositions all target a premature UAG stop codon within the cLuc coding sequence. 293T cells were

transfected with ADAR1 (a and b) or ADAR2 (c and d), luciferase reporter construct and indicated compositions. cLuc activity was measured and normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0028] Figure 6. Provided technologies can provide desired activities in various cell types without exogenous ADAR. Figure 6 depicts editing of an endogenous target (TAG site in 3’ UTR of actin) without exogenous ADAR in different cell types. Cell were transfected with 50 nM oligonucleotides and editing was measured 48 hours later. (N=1 for RPE and NHBE cells, N=2 biological replicates for Hepatocytes)

[0029] Figure 7. Provided technologies comprising various numbers of mismatches can provide desired activities. Compositions all target a premature UAG stop codon within the cLuc coding sequence and have 0-2 mismatches. 293T cells were transfected with ADAR1 or ADAR2, luciferase reporter construct and indicated compositions. cLuc activity was measured at 48 and 96 hrs and normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0030] Figure 8. Provided technologies comprising various patterns of mismatches can provide desired activities. Compositions all target a premature UAG stop codon within the cLuc coding sequence.

293T cells were transfected with ADAR1 or ADAR2, luciferase reporter construct and indicated compositions. cLuc activity was normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0031] Figure 9. Provided technologies comprising various patterns of mismatches can provide desired activities. Compositions all target a premature UAG stop codon within the cLuc coding sequence.

293T cells were transfected with ADAR1 or ADAR2, luciferase reporter construct and indicated compositions. cLuc activity was normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0032] Figure 10. Chirally controlled oligonucleotide compositions can provide desired activities. Compositions all target a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 or ADAR2, luciferase reporter construct and indicated compositions. cLuc activity was normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0033] Figure 11. Chirally controlled oligonucleotide compositions can provide desired activities. Compositions all target a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 or ADAR2, luciferase reporter construct and indicated compositions at varying oligonucleotide concentrations. cLuc activity was normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0034] Figure 12. Chirally controlled oligonucleotide compositions can provide significantly higher activities in various cell types without exogenous ADAR. Compositions all target a UAG motif in the 3’UTR of Actin. Cells were treated gymnotically with oligonucleotides at 10 uM dose, or transfected at 50nM dose. RNA was harvested 48 hours later and percentage of edited transcripts was quantified by Sanger sequencing (n=2 biological replicates).

[0035] Figure 13. Provided technologies can provide desired activities with short sequences without exogenous ADAR. Figure 13 depicts editing in primary human retinal pigmented epithelial (RPE cells). Compositions all target a UAG motif in the 3’UTR of actin. Primary human RPE cells were transfected with 50 nM of oligonucleotides. RNA was harvested 48 hours later and percentage of edited transcripts was quantified by Sanger sequencing (n=2 biological replicates).

[0036] Figure 14. Chirally controlled oligonucleotide compositions can provide high activities in various cell types without exogenous ADAR. Compositions all target a UAG motif in the 3’UTR of actin. Primary human bronchial epithelial cells were treated gymnotically with 10 uM of oligonucleotides, while primary RPE cells were transfected with 50nm of oligonucleotides. RNA was harvested 48 hours later and percentage of edited transcripts was quantified by Sanger sequencing (n=2 biological replicates).

[0037] Figure 15. Provided technologies comprising various internucleotidic linkage patterns can provide desired activities. Compositions all have the same base sequence and target a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 or ADAR2, luciferase reporter construct and indicated compositions. cLuc activity was normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0038] Figure 16. Provided technologies comprising various internucleotidic linkage patterns can provide desired activities without exogenous ADAR. Compositions all target a UAG motif in the 3’UTR of Actin. Primary human hepatocytes were treated gymnotically with 3.3 uM oligonucleotides. RNA was harvested 48 hours later and percentage of edited transcripts was quantified by Sanger sequencing (n=2 biological replicates).

[0039] Figure 17. Provided technologies comprising various modifications and chiral control can provide desired activities without exogenous ADAR. Compositions all target a UAG motif in the 3’UTR of Actin. Primary human hepatocytes were transfected with 50nM of oligonucleotides. RNA was harvested 48 hours later and percentage of edited transcripts was quantified by Sanger sequencing (n=2 biological replicates).

[0040] Figure 18. Provided technologies comprising additional moieties can provide high activities without exogenous ADAR. (a) and (b): Compositions all target an adenosine in the 3’UTR of beta-actin mRNA. Primary human hepatocytes were gymnotically treated at varying concentrations. Editing of target was measured by Sanger sequencing (n=2 biological replicates).

[0041] Figure 19. Provided technologies can provide desired activities without exogenous ADAR. Figure 19 depicts editing of SERPINA1 (PiZ allele) in primary mouse hepatocytes. Compositions all target an adenosine in the mutant human SERPINA1 transcript (PiZZ allele). Primary hepatocytes (extracted from a mouse model expressing the mutant human transcript) were transfected with 50 nM of oligonucleotides. Editing of target was measured by Sanger sequencing (n=2 biological replicates).

[0042] Figure 20. Provided technologies comprising modified bases can provide high activities without exogenous ADAR. Figure 20 depicts editing of SERPINA1 (PiZ allele) in primary mouse hepatocytes. Compositions all target an adenosine in the mutant human SERPINA1 transcript (PiZZ allele). Primary hepatocytes (extracted from a mouse model expressing the mutant human transcript) were transfected treated with 50nM oligonucleotides. Editing of target was measured by Sanger sequencing (n=2 biological replicates). As shown, provided designs comprising modified nucleobases can greatly improve activities.

[0043] Figure 21. Provided technologies comprising modified bases can provide desired activities. Compositions all target a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 or ADAR2, luciferase reporter construct and indicated compositions at varying oligonucleotide concentrations. cLuc activity was normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0044] Figure 22. Figure 22 depicts an example of oligonucleotide configuration.

[0045] Figure 23. Provided technologies comprising modified bases can provide high activities. Compositions all target a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 or ADAR2, luciferase reporter construct and indicated compositions. cLuc activity was normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0046] Figure 24. Provided technologies comprising chirally controlled oligonucleotide compositions can provide high activities compared to stereorandom oligonucleotide compositions. Compositions target UAG motifs within indicated transcripts using endogenous ADAR. Primary human hepatocytes were transfected with compositions at 50 nM oligonucleotide concentration. Percent editing of targeted transcripts was determined through Sanger sequencing of harvested RNA after 48 hour treatment (n = 2 biological replicates). As demonstrated, provided technologies can provide high editing efficiency for various target transcripts.

[0047] Figure 25. Provided technologies comprising oligonucleotides with modified internucleotidic linkages can provide high activities. In some embodiments, provided oligonucleotides comprise phosphorothioate linkages and non-negatively charged internucleotidic linkages such as n001. Compositions target a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1-p150, luciferase reporter construct, and indicated compositions at 3.3 nM oligonucleotide concentrations. cLuc activity was normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0048] Figure 26. Provided technologies comprising oligonucleotides comprising additional chemical moieties can provide high activities. Compositions target an adenosine in the 3’UTR of beta-actin mRNA using endogenous ADAR. Primary monkey hepatocytes were gymnotically treated with indicated compositions at indicated concentrations. Editing of target was measured by Sanger sequencing (n=2 biological replicates).

[0049] Figure 27. Provided technologies comprising oligonucleotides with modified internucleotidic linkages, sugar modifications, and/or additional chemical moieties can provide high activities. Compositions target an adenosine in the 3’UTR of beta-actin mRNA using endogenous ADAR. Primary human hepatocytes were gymnotically treated with indicated compositions at indicated oligonucleotide concentrations. Percent editing of target transcripts was determined through Sanger sequencing (n=2 biological replicates).

[0050] Figure 28. Provided technologies comprising oligonucleotides comprising various modified internucleotidic linkages, sugar modifications and/or additional moieties can provide high activities. Compositions target an adenosine in the 3’UTR of beta-actin mRNA using endogenous ADAR. Primary human hepatocytes were gymnotically treated with indicated compositions at indicated oligonucleotide concentrations. Percent editing of target transcripts was determined through Sanger sequencing (n=2 biological replicates). In some embodiments, certain structural elements, e.g., 2’-F modified sugars in second subdomains, Rp phosphorothioate linkages bonded to second subdomain nucleosides, positioning and/or presence or absence of mismatches, and/or non-negatively charged internucleotidic linkages such as n001 at certain locations can improve editing efficiency.

[0051] Figure 29. Provided technologies comprising oligonucleotides with modified internucleotidic linkages, sugar modifications, and/or additional chemical moieties can provide high activities. Primary human hepatocytes were treated gymnotically with indicated oligonucleotide compositions at indicated concentrations. ADAR was endogenous. Compositions target an adenosine in the 3’ UTR of beta-actin mRNA. Percentage of edited transcripts was quantified by Sanger sequencing (n=2 biological replicates).

[0052] Figure 30. Provided technologies comprising oligonucleotides with modified internucleotidic linkages, sugar modifications, and/or additional chemical moieties can provide high activities. Primary human (a; *: not determined) or monkey (b) hepatocytes were treated gymnotically with indicated oligonucleotide compositions at indicated concentrations. ADAR was endogenous. Compositions target an adenosine in the 3’ UTR of beta-actin mRNA. Percentage of edited transcripts was quantified by Sanger sequencing (n=2 biological replicates).

[0053] Figure 31. Chiral control can improve editing efficiency. Compositions all target a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with plasmids encoding ADAR1-p110 or -p150, luciferase reporter construct and indicated oligonucleotide compositions. cLuc activity was normalized to Gluc expression in mock treated samples (n=2 biological replicates). Among other things, increased numbers/levels of chirally controlled internucleotidic linkages (e.g., Sp phosphorothioate internucleotidic linkages) improved editing efficiency for both ADAR1-p110 and ADAR1-p150.

[0054] Figure 32. Chiral control can improve editing efficiency. Compositions all target a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with plasmids encoding ADAR1-p110 or -p150, luciferase reporter construct and indicated oligonucleotide compositions. cLuc activity was normalized to Gluc expression in mock treated samples (n=2 biological replicates). Among other things, increased numbers/levels of chirally controlled internucleotidic linkages (e.g., Sp phosphorothioate internucleotidic linkages) improved editing efficiency for both ADAR1-p110 and ADAR1-p150.

[0055] Figure 33. Chiral control and modified internucleotidic linkages can improve editing efficiency. (a) Compositions all target a premature UAG stop codon within the cLuc coding sequence.

293T cells were transfected with plasmids encoding ADAR1-p110, luciferase reporter construct and indicated compositions. cLuc activity was normalized to Gluc expression in mock treated samples (n=2 biological replicates). (b) Compositions all target a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with plasmids encoding ADAR1-p150, luciferase reporter construct and indicated compositions. cLuc activity was normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0056] Figure 34. Assessment of oligonucleotides comprising natural phosphate linkages. In some embodiments, natural phosphate linkages can be utilized in accordance with the present disclosure (e.g., numbers, levels, positions, in combination with other structural features (e.g., modifications, patterns, etc.), etc.) to provide oligonucleotide compositions of certain levels of activities. In Figure 34, natural phosphate linkages can be utilized in oligonucleotides in accordance with the present disclosure. Compositions all target a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with plasmids encoding ADAR1-p150 or ADAR2, luciferase reporter construct and indicated compositions. cLuc activity was normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0057] Figure 35. Various sugar modifications may be utilized to provide oligonucleotide compositions with desired activities. Compositions all target a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with plasmids encoding ADAR1-p150 or ADAR2, luciferase reporter construct and indicated compositions. cLuc activity was normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0058] Figure 36. Various sugar modifications may be utilized to provide oligonucleotide compositions with desired activities. (a) Compositions all target a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with plasmids encoding ADAR1-p150 or ADAR2,

luciferase reporter construct and indicated compositions. cLuc activity was normalized to Gluc expression in mock treated samples (n=2 biological replicates). (b) Compositions all target a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with plasmids encoding ADAR1-p150 or ADAR2, luciferase reporter construct and indicated compositions. cLuc activity was normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0059] Figure 37. Provided technologies can provide effective editing in primates. Non-human primates (NHP) were dosed with several compositions (WV-37314, WV-37315, and WV-37330). Compositions all target an adenosine in the 3’UTR of beta-actin mRNA. Animals were dosed subcutaneously once a day for 5 consecutive days (5 mg/kg) with indicated compositions (n=2 animals per composition). Liver biopsy samples were collected two days post last dose. Editing of target was measured by Sanger sequencing. All three compositions administered provided significant levels of in vivo editing of ACTB mRNA (25-50% editing) without administration of exogenous ADAR.

[0060] Figure 38. Provided technologies can provide long-lasting editing activities in vivo.. Provided compositions were assessed in non-human primates (NHP). Certain data for liver (a) and kidney (b) are illustrated. Compositions all target an adenosine in the 3’UTR of beta-actin mRNA. Animals were dosed subcutaneously once a day for 5 consecutive days (5mg/kg) with indicated compositions (n=2 animals per oligonucleotide). Liver biopsy samples were collected 2 days post last dose and 45 days post last dose and kidney biopsy samples were collected at 45 days post last dose. Editing of target was measured by Sanger sequencing. Oligonucleotides in tissues were measured by hybridization ELISA.

[0061] Figure 39. Provided technologies can provide effective editing in various systems including neuronal cells. In some embodiments, compositions were assessed in human iCell neurons and iCell astrocytes (a) and (b), respectively). Compositions all target a UAG motif in the 3’UTR of ACTB and comprise oligonucleotides with the same base sequence. Cells were gymnotically treated with indicated compositions at indicated concentrations and RNA was harvested 6 and 5 days later, respectively. Editing of target was measured by Sanger sequencing (n=2-3 biological replicates). Certain initial results for certain additional targets are presented in (c) and (d). Compositions were assessed in human iCell neurons and iCell astrocytes for editing of 6 different target sites. Each composition targets a UAG motif in the indicated transcript. Cells were gymnotically treated with indicated composition at indicated concentrations, and RNA was harvested 6 days later. Editing of target was measured by Sanger sequencing (n=2 biological replicates).

[0062] Figure 40. Mice engineered to express human ADAR1 can provide editing activity profiles more similar to human cells compared to mice not so engineered. Compositions were assessed in primary hepatocytes harvested from a human ADAR1-transgenic mouse, wild-type mouse and human primary hepatocytes. Oligonucleotides administered comprise GalNAc moieties. Certain data for editing of two

different transcripts, UGP2 (a) and EEF1A1 (b), are shown. For each target, certain data from three compositions of oligonucleotides with identical base sequence but different modifications were presented. Editing of target was measured by Sanger sequencing (n=2-3 biological replicates). As confirmed, in some embodiments chiral control and/or various modifications can be utilized to effectively improve editing levels in accordance with the present disclosure. For each type of cells, from left to right: (a): WV-38701, WV-38700, and WV-38702; (b): WV-38698, WV-38697, and WV-38699.

[0063] Figure 41. Provided technologies can provide editing in vivo. Certain in vivo data, e.g., from livers of human-ADAR1-transgenic mice were presented. Animals were treated with compositions of oligonucleotides comprising GalNAc. Wild-type (WT) mice were included as controls. Certain data for editing of UAG motifs on two different transcripts, UGP2 (a) and EEF1A1 (b), are shown. For each target, certain data from two compositions of oligonucleotides with identical sequence but different modifications were presented. Three animals in each treatment group were dosed with PBS or 10 mg/kg of indicated compositions on days 1, 3, and 5, and liver biopsies were collected on day 8. (n=3 mice per group). As confirmed, in some embodiments chirally controlled oligonucleotide compositions of oligonucleotides comprising non-negatively charged internucleotidic linkages (e.g., n001) can be utilized to effectively improve editing levels, including in vivo, in accordance with the present disclosure.

[0064] Figure 42. Provided technologies can provide editing in vivo in various tissues including in central nervous system. Compositions were assessed in CNS tissues of human-ADAR1-transgenic mice. Certain data for editing of UGP2 (a) and SRSF1 (b) transcripts were presented. Animals were treated with compositions by ICV injection. Five mice in each group were injected with PBS, 2x50 ug doses of oligonucleotide composition on day 0 and 2, or a single 100 ug dose on day 0. Animals were necropsied on day 7. RNA from indicated tissues was harvested, and editing measured by Sanger sequencing (n=5 mice per group). Oligonucleotide distribution in different brain tissue was also measured by hybridization ELISA. Among other things, it was confirmed that provided oligonucleotides can be delivered to various tissues and provide editing activities therein.

[0065] Figure 43. Reduction of certain proteins using siRNA. Shown are ADAR1 p150 (top), ADAR1 p110 (middle) and vinculin loading control (below) in ARPE-19 cells treated with indicated siRNA reagents with or without IFN-a.

[0066] Figure 44. Certain editing data of endogenous ACTB observed with WV-23928 or WV-27395 with siRNA-mediated depletion of the indicated ADAR, with and without IFN-a treatment. N≥3, mean ± SEM. **** P<0.0001 by Welch’s two-way ANOVA followed by two-tailed post-hoc test. nd, not detected; NTC non-targeting control.

[0067] Figure 45. Figure 45. Provided technologies can provide highly specific editing. (a) Scatter

plot (top) of variants detected in WV-30298 samples. On-target ACTB editing and off-target edits have >3 LOD score and >5% editing. LOD score calculated by Mutect2 indicates the likelihood odds ratio that a variant exists in treated samples compared with mock samples. Genes with the highest percentage editing and highest LOD scores are labeled. Total RNA coverage (bottom) across replicates for all variants (potential edit sites). (b) Scatter plot (top) of variants and total RNA coverage (bottom) in WV-27458 samples.

[0068] Figure 46. Provided technologies can provide multiplex editing. Presented are certain data in primary human hepatocytes. (a) Percentage editing observed on indicated transcripts in the presence of 20 nM each of a single (Isolated) or multiple (Multiplex) oligonucleotide compositions after transfection of primary human hepatocytes. (b) Percentage editing detected on indicated transcripts in the presence of 1.1 uM each of a single (Isolated) or multiple (Multiplex) GalNAc-conjugated oligonucleotides. N=3, mean ± SEM. * P<0.05, *** P<0.001 by two-tailed Welch’s t-test. For each target, from left to right: Isolated, Multiplex.

[0069] Figure 47. Mice engineered to express human ADAR1 can provide editing activity profiles more similar to human cells compared to mice not so engineered. Compositions were assessed in primary hepatocytes harvested from a human ADAR1-transgenic mouse, wild-type mouse and human primary hepatocytes. Oligonucleotides administered comprise GalNAc moieties. Certain data for editing of two different transcripts, UGP2 (a-c) and EEF1A1 (d-f), are shown. For each target, certain data from three compositions of oligonucleotides with identical base sequence but different modifications were presented. Editing of target was measured by Sanger sequencing (n=2-3 biological replicates). As confirmed, in some embodiments chiral control and/or various modifications can be utilized to effectively improve editing levels in accordance with the present disclosure.

[0070] Figure 48. Provided technologies provide editing in various cell types including CD8+ T cells. Figure 48 depicts editing in primary human CD8+ T cells by gymnotic uptake. All oligonucleotides have the same sequence and all target a UAG motif in the 3’UTR of ACTB. Primary T cells were pre-stimulated for 24 or 96 hrs as indicated and then were gymnotically treated with oligonucleotide compositions at indicated concentrations, and RNA was harvested 4 days later. Editing of target was measured by Sanger sequencing (n=2 biological replicates).

[0071] Figure 49. Provided technologies provide editing in various cell types including primary human fibroblasts. Figure 49 depicts certain editing in primary human fibroblasts by gymnotic uptake and transfection. The oligonucleotide composition WV-37318 targets a UAG motif in the 3’UTR of ACTB. Three different primary human fibroblast lines were treated by transfection (50 nM) or by gymnotic uptake (10 uM) as indicated, and RNA was harvested 60 hours later. Editing of target was measured by Sanger sequencing (n=2 biological replicates).

[0072] Figure 50. Provided technologies provide editing in various cell types including in ex vivo retinal tissue isolated from non-human primate eyes. Figure 50 depicts certain editing in ex vivo retinal tissue isolated from non-human primate eyes. The oligonucleotide composition targets a UAG motif in the 3’UTR of ACTB. In two independent experiments, eyeballs from NHPs were freshly dissected and retinal tissue was treated with oligonucleotide composition by gymnotic uptake. RNA was harvested 48 hours later. Editing of target was measured by Sanger sequencing (n=4-5 biological replicates per experimental condition).

[0073] Figure 51. Provided technologies comprising various modifications can provide editing. Figure 51 ((a)-(d)) depicts certain editing in primary human hepatocytes. The oligonucleotide compositions target specific adenosine residues (surrogate site#1, 2, 3, or 4) in a coding sequence of a wild-type SERPINA1 (SA1) transcript. As confirmed, oligonucleotides comprising various modifications, sequences and/or additional chemical moieties can provide desired editing. Primary human hepatocytes were treated with indicated compositions and concentrations. RNA was harvested 48 hours later. Editing of target was measured by Sanger sequencing (n=2 biological replicates).

[0074] Figure 52. Provided technologies comprising various modifications can provide editing. Figure 52 depicts certain editing in primary human hepatocytes. The oligonucleotide compositions target specific adenosine residues (surrogate site#1, 2, 3, or 4) in a coding sequence of a wild-type SERPINA1 (SA1) transcript. Primary human hepatocytes were treated with indicated oligonucleotide compositions and concentrations. RNA was harvested 48 hours later. Editing of target was measured by Sanger sequencing (n=2 biological replicates).

[0075] Figure 53. Provided technologies comprising various modifications can provide editing. Figure 53, (a), depicts editing in primary human hepatocytes. The oligonucleotide compositions target a specific adenosine residue (surrogate site#1) in a coding sequence of a wild-type SERPINA1 (SA1) transcript. Primary human hepatocytes were treated with indicated oligonucleotide compositions and concentrations. RNA was harvested 48 hours later. Editing of target was measured by Sanger sequencing (n=2 biological replicates). Figure 53, (b) depicts editing in primary human hepatocytes. The oligonucleotide compositions target a specific adenosine residue (surrogate site#2) in a coding sequence of a wild-type SERPINA1 (SA1) transcript. Primary human hepatocytes were treated with indicated oligonucleotide compositions and concentrations. RNA was harvested 48 hours later. Editing of target was measured by Sanger sequencing (n=2 biological replicates). Figure 53, (c), depicts editing in primary human hepatocytes. The oligonucleotide compositions target a specific adenosine residue (surrogate site#1 or #2) in a coding sequence of a wild-type SERPINA1 (SA1) transcript. Primary human hepatocytes were treated with indicated oligonucleotide compositions and concentrations. RNA was harvested 48 hours later. Editing of target was measured by Sanger sequencing (n=2 biological replicates).

[0076] Figure 54. Removing wobbles and/or mismatches may improve editing levels. Figure 54 depicts editing in primary human and NHP hepatocytes. The oligonucleotide compositions target a specific adenosine residue (surrogate site#1 or #2) in a coding sequence of a WT SERPINA1 (SA1) transcript. Primary human and NHP hepatocytes were treated with indicated oligonucleotide compositions and concentrations. RNA was harvested 48 hours later. Editing of target was measured by Sanger sequencing (n=2 biological replicates).

[0077] Figure 55. Provide technologies can provide editing in NHP and human cells at various concentrations. Figure 55 depicts editing in primary human and NHP hepatocytes. The oligonucleotide compositions target a specific adenosine residue (surrogate site#1 or #2) in a coding sequence of a wild-type SERPINA1 (SA1) transcript. Both oligonucleotide compositions have a G-U wobble against the NHP mRNA sequence. Primary human and NHP hepatocytes were treated with indicated oligonucleotide compositions and concentrations. RNA was harvested 48 hours later. Editing of target was measured by Sanger sequencing (n=2 biological replicates).

[0078] Figure 56. Provide technologies comprising various modifications can provide editing. Figure 56 depicts editing in primary NHP hepatocytes. The oligonucleotide compositions target a specific adenosine residue (surrogate site#2) in a coding sequence of a wild-type SERPINA1 (SA1) transcript. Primary NHP hepatocytes were treated with indicated oligonucleotide compositions and concentrations. RNA was harvested 48 hours later. Editing of target was measured by Sanger sequencing (n=2 biological replicates).

[0079] Figure 57. Provide technologies comprising various modifications including base modifications can provide editing. The oligonucleotide compositions all target a premature UAG stop codon within a cLuc coding sequence. 293T cells were transfected with ADAR-p110 or ADAR1-p150, luciferase reporter construct and indicated oligonucleotide compositions. cLuc activity was measured and normalized to Gluc expression in mock treated samples (n=2 biological replicates).

[0080] Figure 58. Provide technologies comprising various modifications including abasic units can provide editing. The oligonucleotide compositions all target a premature UAG stop codon within a cLuc coding sequence. 293T cells were transfected with ADAR-p110 or ADAR1-p150, luciferase reporter construct and indicated oligonucleotide compositions. cLuc activity was measured and normalized to Gluc expression in mock treated samples (n=2 biological replicates).

CLAIMS

1. An oligonucleotide comprising:

a first domain; and

a second domain,

wherein:

the first domain comprises one or more 2’-F modifications;

the second domain comprises one or more sugars that do not have a 2’-F modification.

2. An oligonucleotide comprising one or more modified sugars and/or one or more modified internucleotidic linkages, wherein the oligonucleotide comprises a first domain and a second domain each independently comprising one or more nucleobases.

3. The oligonucleotide of claim 1, wherein when the oligonucleotide is contacted with a target nucleic acid comprising a target adenosine in a system, a target adenosine in the target nucleic acid is modified, and the modification is or comprises conversion of the target adenosine to an inosine.

4. The oligonucleotide of claim 3, wherein the modification is promoted by an ADAR protein. 5. The oligonucleotide of claim 4, wherein the oligonucleotide has a length of about 26-35 nucleobases.

6. The oligonucleotide of claim 4, wherein the second domain has a length of about 10-50 nucleobases.

7. The oligonucleotide of claim 3, wherein about 50%-100% of sugars in the first domain independently comprise a 2’-F modification.

8. The oligonucleotide of claim 6, wherein about 50%-100% of internucleotidic linkages in the first domain are modified internucleotidic linkages.

9. The oligonucleotide of claim 7, wherein at least about 1-50 chiral internucleotidic linkages in the first domain is chirally controlled.

10. The oligonucleotide of claim 8, wherein the first domain comprises one or more phosphorothioate internucleotidic linkages.

11. The oligonucleotide of claim 9, wherein the first domain comprises 1, 2, 3, 4, or 5 non-negatively charged internucleotidic linkages.

12. The oligonucleotide of claim 10, wherein the internucleotidic linkage between the first and the second nucleosides of the first domain is a non-negatively charged internucleotidic linkage.

13. The oligonucleotide of claim 9, wherein the second domain has a length of about 10-50 nucleobases.

14. The oligonucleotide of claim 13, wherein the second domain comprise a nucleoside opposite to a target adenosine when the oligonucleotide is aligned with a target nucleic acid for complementarity.

15. The oligonucleotide of claim 14, wherein the opposite nucleobase is optionally substituted or protected U, or is an optionally substituted or protected tautomer of U, or is optionally substituted or protected C, or is an optionally substituted or protected tautomer of C, or is optionally substituted or protected A, or is an optionally substituted or protected tautomer of A, or is optionally substituted or protected nucleobase of pseudoisocytosine, or is an optionally substituted or protected tautomer of the nucleobase of pseudoisocytosine, or is a nucleobase BA, wherein BA is or comprises Ring BA or a tautomer thereof, wherein Ring BA is an optionally substituted, 5-20 membered, monocyclic, bicyclic or polycyclic ring having 0-10 hetereoatoms.

16. The oligonucleotide of claim 15, wherein the nucleobase is BA, wherein BA is or comprises Ring BA or a tautomer thereof, wherein Ring BA is an optionally substituted, 5-20 membered, monocyclic, bicyclic or polycyclic ring having 0-10 hetereoatoms.

17. The oligonucleotide of claim 16, wherein BA has weaker hydrogen bonding with the target adenine of the adenosine compared to U.

18. The oligonucleotide of claim 16, wherein Ring BA comprises X2 X3 , X2 X3 X4 , −X1( ) X2 X3 , −X1( ) X2 X3 X4 , or has the structure of formula BA-I, BA-I-a, BA-I-b, BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a or BA-III-b.

19. The oligonucleotide of claim 14, wherein the opposite nucleobase is

20. The oligonucleotide of claim 14, wherein the opposite nucleobase is

21. The oligonucleotide of claim 14, wherein the opposite nucleobase is

22. The oligonucleotide of claim 14, wherein about 50%-100% of sugars in the second domain are independently modified sugars with a modification that is not 2’-F.

23. The oligonucleotide of claim 22, wherein about 50%-100% of internucleotidic linkages in the second domain are modified internucleotidic linkages.

24. The oligonucleotide of claim 23, wherein each modified internucleotidic linkages is independently a phosphorothioate internucleotidic linkage or a non-negatively charged internucleotidic linkage.

25. The oligonucleotide of claim 24, wherein the second domain comprises one or more phosphorothioate internucleotidic linkages.

26. The oligonucleotide of claim 25, wherein the second domain comprises 1, 2, 3, 4, or 5 non-negatively charged internucleotidic linkages.

27. The oligonucleotide of claim 26, wherein the internucleotidic linkage between the last and the second last nucleosides of the second domain is a non-negatively charged internucleotidic linkage.

28. The oligonucleotide of claim 25, wherein at least 50%-100% of chiral internucleotidic linkages in the second domain is chirally controlled.

29. The oligonucleotide of claim 28, wherein the second domain comprises or consists of from the 5’ to 3’ a first subdomain, a second subdomain , and a third subdomain.

30. The oligonucleotide of claim 29, wherein the first subdomain has a length of about 5-50 nucleobases.

31. The oligonucleotide of claim 30, wherein about 50%-100% of sugars in the first subdomain are independently modified sugars with a modification that is not 2’-F.

32. The oligonucleotide of claim 31, wherein the second subdomain has a length of 3 nucleobases.

33. The oligonucleotide of claim 32, wherein the second subdomain comprises a nucleoside opposite to a target adenosine.

34. The oligonucleotide of claim 33, wherein the second subdomain comprises one or more natural DNA sugars.

35. The oligonucleotide of claim 34, wherein the second subdomain comprises one or more natural RNA sugars.

36. The oligonucleotide of claim 34, wherein the second subdomain comprises about a 2’-F modified sugars.

37. The oligonucleotide of claim 34, wherein the sugar of the opposite nucleoside comprises a 2’-OH.

38. The oligonucleotide of claim 34, wherein the sugar of the opposite nucleoside is a natural DNA sugar.

39. The oligonucleotide of claim 34, wherein the sugar of a nucleoside 5’-next to the opposite nucleoside (sugar of N1 in 5’-…N1N0…3’, wherein when aligned with a target, N0 is opposite to a target adenosine) is a natural DNA sugar.

40. The oligonucleotide of claim 34, wherein the sugar of a nucleoside 5’-next to the opposite nucleoside (sugar of N1 in 5’-…N1N0…3’, wherein when aligned with a target, N0 is opposite to a target adenosine) comprises 2’-F.

41. The oligonucleotide of claim 34, wherein the sugar of a nucleoside 3’-next to the opposite nucleoside (sugar of N−1 in 5’-…N0N−1…3’, wherein when aligned with a target, N0 is opposite to a target adenosine) is a natural DNA sugar.

42. The oligonucleotide of claim 34, wherein each of the sugar of the opposite nucleoside, the sugar of a nucleoside 5’-next to the opposite nucleoside (sugar of N1 in 5’-…N1N0…3’, wherein when aligned with a target, N0 is opposite to a target adenosine), and the sugar of a nucleoside 3’-next to the opposite nucleoside (sugar of N−1 in 5’-…N0N−1…3’, wherein when aligned with a target, N0 is opposite to a target adenosine) is independently a natural DNA sugar.

43. The oligonucleotide of claim 34, wherein the sugar of the opposite nucleoside is a natural DNA sugar, the sugar of a nucleoside 5’-next to the opposite nucleoside (sugar of N1 in 5’-…N1N0…3’, wherein when aligned with a target, N0 is opposite to a target adenosine) is a 2’-F modified sugar, and the sugar of a nucleoside 3’-next to the opposite nucleoside (sugar of N−1 in 5’-…N0N−1…3’, wherein when aligned with a target, N0 is opposite to a target adenosine) is a natural DNA sugar.

44. The oligonucleotide of claim 34, wherein the nucleoside opposite to a target nucleoside is connected to its 3’ immediate nucleoside through a Rp phosphorothioate internucleotidic linkage.

45. The oligonucleotide of claim 34, wherein the nucleoside (position -1) that is 3’ immediate to an nucleoside opposite to a target nucleoside (position 0) is connected to its 3’ immediate nucleoside (position -2) through a non-negatively charged internucleotidic linkage.

46. The oligonucleotide of claim 34, wherein the 3’-immediate nucleoside comprises a base that is

not G.

47. The oligonucleotide of claim 34, wherein the 3’-immediate nucleoside comprises hypoxanthine.

48. The oligonucleotide of claim 34, wherein the third subdomain has a length of about 1-10 nucleobases.

49. The oligonucleotide of claim 34, wherein the oligonucleotide comprises a moiety that is or comprises GalNAc or a derivative thereof.

50. An oligonucleotide comprising a modified nucleobase as described herein.

51. An oligonucleotide, wherein the oligonucleotide is otherwise identical to an oligonucleotide of any one of the preceding claims, except that at a position of a modified internucleotidic linkage is a linkage having the structure of −O5−PL(RCA)−O3−, wherein:

PL is P, or P(=W);

W is O, S, or WN;

RCA is or comprises an optionally substituted or capped chiral auxiliary moiety,

O5 is an oxygen bonded to a 5’-carbon of a sugar, and

O3 is an oxygen bonded to a 3’-carbon of a sugar.

52. The oligonucleotide of claim 51, wherein at each position of a modified internucleotidic linkage is independently a linkage having the structure of −O5−PL(W)(RCA)−O3−.

53. The oligonucleotide of claim 52, wherein each RCA is independently

, wherein RC1 is R, −Si(R)3 or −SO2R, RC2 and RC3 are taken together with their intervening atoms to form an optionally substituted 3-7 membered saturated or partially unsaturated ring having, in addition to the nitrogen atom, 0-2 heteroatoms, RC4 is −H or −C(O)R’.

54. The oligonucleotide of claim 52, wherein each RCA is independently

55. The oligonucleotide of claim 54, wherein RC1 is −SiPh2Me.

56. The oligonucleotide of claim 54, wherein RC1 is −SO2R, wherein R is optionally substituted phenyl.

57. The oligonucleotide of any one of claims 1-56, wherein the oligonucleotide has a purity of about 10%-100%.

58. A pharmaceutical composition which comprises or delivers an effective amount of an oligonucleotide of any one of claims 1-56 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

59. An oligonucleotide composition comprising a plurality of oligonucleotides, wherein oligonucleotides of the plurality share:

1) a common base sequence, and

2) the same linkage phosphorus stereochemistry independently at one or more chiral internucleotidic linkages (“chirally controlled internucleotidic linkages”);

wherein each oligonucleotide of the plurality is independently an oligonucleotide of any one of claims 1-56 or an acid, base, or salt form thereof; or

an oligonucleotide composition comprising one or more pluralities of oligonucleotides, wherein oligonucleotides of each plurality independently share:

1) a common base sequence, and

2) the same linkage phosphorus stereochemistry independently at one or more chiral internucleotidic linkages (“chirally controlled internucleotidic linkages”);

wherein each oligonucleotide of the plurality is independently an oligonucleotide of any one of claims 1-56 or an acid, base, or salt form thereof; or

a composition comprising a plurality of oligonucleotides which are of a particular oligonucleotide type characterized by:

a) a common base sequence;

b) a common pattern of backbone linkages;

c) a common pattern of backbone chiral centers;

d) a common pattern of backbone phosphorus modifications;

which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same common base sequence, pattern of backbone linkages and pattern of backbone phosphorus modifications, for oligonucleotides of the particular oligonucleotide type, or a non-random level of all oligonucleotides in the composition that share the common base sequence are oligonucleotides of the plurality; and

wherein each oligonucleotide of the plurality is independently an oligonucleotide of any one of claims 1-56 or an acid, base, or salt form thereof.

60. An oligonucleotide composition comprising a plurality of oligonucleotides, wherein oligonucleotides of the plurality share:

1) a common base sequence, and

2) the same linkage phosphorus stereochemistry independently at one or more chiral internucleotidic linkages (“chirally controlled internucleotidic linkages”);

wherein the common base sequence is complementary to a base sequence of a portion of a nucleic acid which portion comprises a target adenosine; or

an oligonucleotide composition comprising one or more pluralities of oligonucleotides, wherein oligonucleotides of each plurality independently share:

1) a common base sequence, and

2) the same linkage phosphorus stereochemistry independently at one or more chiral internucleotidic linkages (“chirally controlled internucleotidic linkages”);

wherein the common base sequence of each plurality is independently complementary to a base sequence of a portion of a nucleic acid which portion comprises a target adenosine; or

a composition comprising a plurality of oligonucleotides which are of a particular oligonucleotide type characterized by:

a) a common base sequence;

b) a common pattern of backbone linkages;

c) a common pattern of backbone chiral centers;

d) a common pattern of backbone phosphorus modifications;

which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same common base sequence, pattern of backbone linkages and pattern of backbone phosphorus modifications, for oligonucleotides of the particular oligonucleotide type, or a non-random level of all oligonucleotides in the composition that share the common base sequence are oligonucleotides of the plurality; and

wherein the common base sequence is complementary to a base sequence of a portion of a nucleic acid which portion comprises a target adenosine.

61. The composition of any one of claims 59-60, wherein the level of oligonucleotides of a plurality in oligonucleotides in the composition that share the common base sequence of the plurality 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 chirally controlled internucleotidic linkages.

62. A phosphoramidite, wherein the nucleobase of the phosphoramidite is a nucleobase as described herein or a tautomer thereof, wherein the nucleobase or tautomer thereof is optionally substituted or protected.

63. A phosphoramidite, wherein the nucleobase is or comprises Ring BA, wherein Ring BA has the

structure of BA-I, BA-I-a, BA-I-b, BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a, BA-III-b, BA-IV, BA-IV- a, BA-IV-b, BA-V, BA-V-a, BA-V-b, or BA-VI, or a tautomer of Ring BA, wherein the nucleobase is optionally substituted or protected.

64. The phosphoramidite of claim 62 or 63, wherein the phosphoramidite has the structure of RNS−P(OR)N(R)2, wherein RNS is a optionally protected nucleoside moiety, and each R is as described herein.

65. The phosphoramidite of claim 64, wherein the phosphoramidite has the structure of RNS−P(OCH2CH2CN)N(i-Pr)2.

66. The phosphoramidite of claim 62 or 63, wherein the phosphoramidite comprises a chiral auxiliary moiety, wherein the phosphorus is bonded to an oxygen and a nitrogen atom of the chiral auxiliary moiety.

67. The phosphoramidite of claim 62 or 63, wherein the phosphoramidite has the structure of

68. The phosphoramidite of claim 67, wherein RC1 is −SiPh2Me.

69. The phosphoramidite of claim 67, wherein RC1 is −SO2R, wherein R is optionally substituted C1-10 aliphatic or wherein R is optionally substituted phenyl.

70. A method for preparing an oligonucleotide or composition, comprising coupling a 5’-OH of an oligonucleotide or a nucleoside with a phosphoramidite of any one of claims 62-69.

71. The method of claim 70, wherein the oligonucleotide, or an oligonucleotide in the composition, comprises a sugar comprising 2’-OH.

72. A method for characterizing an oligonucleotide or a composition, comprising:

administering the oligonucleotide or composition to a cell or a population thereof comprising or expressing an ADAR1 polypeptide or a characteristic portion thereof, or a polynucleotide encoding an ADAR1 polypeptide or a characteristic portion thereof; or

administering the oligonucleotide or composition to a non-human animal or a population thereof comprising or expressing an ADAR1 polypeptide or a characteristic portion thereof, or a polynucleotide encoding an ADAR1 polypeptide or a characteristic portion thereof.

73. A method for modifying a target adenosine in a target nucleic acid, comprising contacting the target nucleic acid with an oligonucleotide or composition of any one of the preceding claims; or 74. a method for deaminating a target adenosine in a target nucleic acid, comprising contacting the target nucleic acid with an oligonucleotide or composition of any one of the preceding claims; or

a method for producing, or restoring or increasing level of a product of a particular nucleic acid, comprising contacting a target nucleic acid with an oligonucleotide or composition of any one of the preceding claims, wherein the target nucleic acid comprises a target adenosine, and the particular nucleic acid differs from the target nucleic acid in that the particular nucleic acid has an I or G instead of the target adenosine; or

a method for reducing level of a product of a target nucleic acid, comprising contacting a target nucleic acid with an oligonucleotide or composition of any one of the preceding claims, wherein the target nucleic acid comprises a target adenosine; or

a method, comprising:

contacting an oligonucleotide or composition of any one of the preceding claims with a sample comprising a target nucleic acid and an adenosine deaminase, wherein:

the base sequence of the oligonucleotide or oligonucleotides in the oligonucleotide composition is substantially complementary to that of the target nucleic acid; and

the target nucleic acid comprises a target adenosine;

wherein the target adenosine is modified; or

a method, comprising

1) obtaining a first level of modification of a target adenosine in a target nucleic acid, which level is observed when a first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and an adenosine deaminase, wherein the first oligonucleotide composition comprises a first plurality of oligonucleotides sharing the same base sequence which is substantially complementary to that of the target nucleic acid; and

2) obtaining a reference level of modification of a target adenosine in a target nucleic acid, which level is observed when a reference oligonucleotide composition is contacted with a sample comprising the target nucleic acid and an adenosine deaminase, wherein the reference oligonucleotide composition comprises a reference plurality of oligonucleotides sharing the same base sequence which is substantially complementary to that of the target nucleic acid;

wherein:

oligonucleotides of the first plurality comprise more sugars with 2’-F modification, more sugars with 2’-OR modification wherein R is not −H, and/or more chiral internucleotidic linkages than oligonucleotides of the reference plurality; and

the first oligonucleotide composition provides a higher level of modification compared to oligonucleotides of the reference oligonucleotide composition; or

a method, comprising

obtaining a first level of modification of a target adenosine in a target nucleic acid, which level is observed when a first oligonucleotide composition is contacted with a sample comprising the target

nucleic acid and an adenosine deaminase, wherein the first oligonucleotide composition comprises a first plurality of oligonucleotides sharing the same base sequence which is substantially complementary to that of the target nucleic acid; and

wherein the first level of modification of a target adenosine is higher than a reference level of modification of the target adenosine, wherein the reference level is observed when a reference oligonucleotide composition is contacted with a sample comprising the target nucleic acid and an adenosine deaminase, wherein the reference oligonucleotide composition comprises a reference plurality of oligonucleotides sharing the same base sequence which is substantially complementary to that of the target nucleic acid;

wherein:

oligonucleotides of the first plurality comprise more sugars with 2’-F modification, more sugars with 2’-OR modification wherein R is not −H, and/or more chiral internucleotidic linkages than oligonucleotides of the reference plurality; or

a method, comprising

1) obtaining a first level of modification of a target adenosine in a target nucleic acid, which level is observed when a first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and an adenosine deaminase, wherein the first oligonucleotide composition comprises a first plurality of oligonucleotides sharing the same base sequence which is substantially complementary to that of the target nucleic acid; and

2) obtaining a reference level of modification of a target adenosine in a target nucleic acid, which level is observed when a reference oligonucleotide composition is contacted with a sample comprising the target nucleic acid and an adenosine deaminase, wherein the reference oligonucleotide composition comprises a reference plurality of oligonucleotides sharing the same base sequence which is substantially complementary to that of the target nucleic acid;

wherein:

oligonucleotides of the first plurality comprise more sugars with 2’-F modification, more sugars with 2’-OR modification wherein R is not −H, and/or more chirally controlled chiral internucleotidic linkages than oligonucleotides of the reference plurality; and

the first oligonucleotide composition provides a higher level of modification compared to oligonucleotides of the reference oligonucleotide composition; or

a method, comprising

obtaining a first level of modification of a target adenosine in a target nucleic acid, which level is observed when a first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and an adenosine deaminase, wherein the first oligonucleotide composition comprises a first plurality of oligonucleotides sharing the same base sequence which is substantially complementary to that of the target nucleic acid; and

wherein the first level of modification of a target adenosine is higher than a reference level of modification of the target adenosine, wherein the reference level is observed when a reference oligonucleotide composition is contacted with a sample comprising the target nucleic acid and an adenosine deaminase, wherein the reference oligonucleotide composition comprises a reference plurality of oligonucleotides sharing the same base sequence which is substantially complementary to that of the target nucleic acid;

wherein:

oligonucleotides of the first plurality comprise more sugars with 2’-F modification, more sugars with 2’-OR modification wherein R is not −H, and/or more chirally controlled chiral internucleotidic linkages than oligonucleotides of the reference plurality; or

a method, comprising

1) obtaining a first level of modification of a target adenosine in a target nucleic acid, which level is observed when a first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and an adenosine deaminase, wherein the first oligonucleotide composition comprises a first plurality of oligonucleotides sharing the same base sequence which is substantially complementary to that of the target nucleic acid; and

2) obtaining a reference level of modification of a target adenosine in a target nucleic acid, which level is observed when a reference oligonucleotide composition is contacted with a sample comprising the target nucleic acid and an adenosine deaminase, wherein the reference oligonucleotide composition comprises a reference plurality of oligonucleotides sharing the same base sequence which is substantially complementary to that of the target nucleic acid;

wherein:

oligonucleotides of the first plurality comprise one or more chirally controlled chiral internucleotidic linkages; and

oligonucleotides of the reference plurality comprise no chirally controlled chiral internucleotidic linkages (a reference oligonucleotide composition is a “stereorandom composition); and

the first oligonucleotide composition provides a higher level of modification compared to oligonucleotides of the reference oligonucleotide composition; or

a method, comprising

obtaining a first level of modification of a target adenosine in a target nucleic acid, which level is observed when a first oligonucleotide composition is contacted with a sample comprising the target nucleic acid and an adenosine deaminase, wherein the first oligonucleotide composition comprises a first plurality of oligonucleotides sharing the same base sequence which is substantially complementary to that of the target nucleic acid; and

wherein the first level of modification of a target adenosine is higher than a reference level of modification of the target adenosine, wherein the reference level is observed when a reference oligonucleotide composition is contacted with a sample comprising the target nucleic acid and an adenosine deaminase, wherein the reference oligonucleotide composition comprises a reference plurality of oligonucleotides sharing the same base sequence which is substantially complementary to that of the target nucleic acid;

wherein:

oligonucleotides of the first plurality comprise one or more chirally controlled chiral internucleotidic linkages; and

oligonucleotides of the reference plurality comprise no chirally controlled chiral internucleotidic linkages (a reference oligonucleotide composition is a “stereorandom composition).

75. The method of claim 74, wherein a first oligonucleotide composition is an oligonucleotide composition of any one of the preceding claims.

76. The method of claim 70-75, wherein the deaminase is an ADAR enzyme.

77. The method of claim 76, wherein the target nucleic acid is more associated with a condition, disorder or disease, or decrease of a desired property or function, or increase of an undesired property or function, compared to a nucleic acid which differs from the target nucleic acid in that it has an I or G at the position of the target adenosine instead of the target adenosine.

78. The method of claim 74, wherein the target adenosine is a G to A mutation.

79. A method for preventing or treating a condition, disorder or disease amenable to a G to A mutation, comprising administering to a subject susceptible thereto or suffering therefrom an effective amount of an oligonucleotide or composition of any one of the preceding claims; or

a method for preventing or treating a condition, disorder or disease associated with a G to A mutation, comprising administering to a subject susceptible thereto or suffering therefrom an effective amount of an oligonucleotide or composition of any one of the preceding claims.

80. The method of any one of claim 79, wherein the condition, disorder or disease is amenable to an A to G or A to I modification.

81. A compound, oligonucleotide, composition or method of the specification or any one of Embodiments 1-889.