Oligonucleotide compositions and methods thereof
Among other things, the present disclosure provides technologies for altering splicing, particularly for increasing inclusion of exons in splicing products. In some embodiments, the present disclosure provides SMN2 oligonucleotides, compositions, and methods thereof. In some embodiments, the present disclosure provides chirally controlled SMN2 oligonucleotide compositions. In some embodiments, provided oligonucleotides and compositions can increase level of an exon 7-containing SMN2 splicing product and/or a gene product thereof. In some embodiments, the present disclosure provides methods for treatment of splicing-related conditions, disorders and diseases. In some embodiments, the present disclosure provides methods for treating SMN2-related conditions, disorders and diseases such as SMA (spinal muscular atrophy) and ALS (amyotrophic lateral sclerosis).
1. A composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:
1) base sequence, wherein the base sequence comprises 15 contiguous bases of TCACTTTCATAATGCTGG (SEQ ID NO: 479), wherein each T can be independently substituted with U;
2) Pattern of backbone linkages;
3) Pattern of backbone chiral centers; and
4) pattern of backbone phosphorus modifications,
oligonucleotides of the plurality comprise at least 5 chiral modified internucleotidic linkages each independently having a stereopurity of at least 90% at its chiral linkage phosphorus; and
the pattern of backbone chiral centers is or comprises:
(Rp/Op)t[(Np/Op)n]y(Rp/Op)m, wherein each of (Rp/Op)t and (Rp/Op)m independently comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Rp;
(Rp)(Rp/Op)t[(Np/Op)n]y(Rp/Op)m(Rp); or
(Rp)t[(Np/Op)n]y(Rp)m;
wherein:
each Np is independently Rp or Sp,
Sp indicates S configuration of a chiral linkage phosphorus of a chiral modified internucleotidic linkage,
Rp indicates R configuration of a chiral linkage phosphorus of a chiral modified internucleotidic linkage,
Op indicates an achiral linkage phosphorus of a natural phosphate linkage,
each of t, n, y, and m is independently 1-50.
2. The composition of claim 1 , wherein the pattern of backbone chiral centers is or comprises (Rp)t[(Sp)n]y(Rp)m, wherein t is 4 or more, n is 1, 2, 3, 4, or 5, y is 1, and m is 4 or more.
3. The composition of claim 2 , wherein the composition is a chirally controlled oligonucleotide composition characterized in that, when it is contacted with a transcript in a SMN2 transcript splicing system, inclusion of exon 7 of SMN2 is increased by 2 fold or more relative to that observed under a reference condition which is absence of the composition.
4. The composition of claim 2 , wherein the composition is a chirally controlled oligonucleotide composition characterized in that, when it is contacted with a transcript in a SMN2 transcript splicing system, inclusion of exon 7 of SMN2 is increased by 2 fold or more relative to that observed under a reference condition which is presence of composition of nusinersen: Teo*m5Ceo*Aeo*m5Ceo*Teo*Teo*Teo*m5Ceo*Aeo*Teo*Aeo*Aeo*Teo*Geo*m5Ceo*Teo*Geo*Geo (SEQ ID NO: 199), wherein each * independently represents a phosphorothioate internucleotidic linkage, each m5Ceo independently represents a 5-methyl 2′-O-methoxyethyl C, and each eo independently represents a 2′-O-(2-methoxyethyl) modification.
5. The composition of claim 1 , wherein each oligonucleotide of the plurality independently comprises an additional chemical moiety capable of binding to the asialoglycoprotein receptor.
6. The composition of claim 5 , wherein the additional chemical moiety is or comprises GalNAc.
7. The composition of claim 1 , wherein the base sequence comprises TCACTTTCATAATGCTGG (SEQ ID NO: 479), wherein each T can be independently substituted with U.
8. The composition of claim 1 , wherein the base sequence is TCACTTTCATAATGCTGG (SEQ ID NO: 1).
9. The composition of claim 1 , wherein the pattern of backbone chiral centers comprises at least 50% Rp.
10. The composition of claim 1 , wherein the pattern of backbone chiral centers comprises at least 70% Rp.
11. The composition of claim 1 , wherein oligonucleotides of the particular oligonucleotide type comprise at least 12 chiral modified internucleotidic linkages in the Rp configuration.
12. The composition of claim 1 , wherein each oligonucleotide of the plurality is independently Teo *R m5Ceo *R Aeo *R m5Ceo *R Teo *R Teo *R Teo *R m5Ceo *R Aeo *S Teo *R Aeo *R Aeo *R Teo *R Geo *R m5Ceo *R Teo *R Geo *R Geo (SEQ ID NO: 213) or a pharmaceutically acceptable salt thereof,
wherein each *R independently represents a phosphorothioate internucleotidic linkage in the Rp configuration, each *S independently represents a phosphorothioate internucleotidic linkage in the Sp configuration, each m5Ceo independently represents a 5-methyl 2′-O-methoxyethyl C, and each eo independently represents a 2′-O-(2-methoxyethyl) modification.
13. The composition of claim 1 , wherein each oligonucleotide of the plurality is independently Teo *R m5Ceo *R Aeo *R m5Ceo *R Teo *R Teo *R Teo *R m5Ceo *S Aeo *S Teo *S Aeo *R Aeo *R Teo *R Geo *R m5Ceo *R Teo *R Geo *R Geo (SEQ ID NO: 214) or a pharmaceutically acceptable salt thereof,
wherein each *R independently represents a phosphorothioate internucleotidic linkage in the Rp configuration, each *S independently represents a phosphorothioate internucleotidic linkage in the Sp configuration, each m5Ceo independently represents a 5-methyl 2′-O-methoxyethyl C, and each eo independently represents a 2′-O-(2-methoxyethyl) modification.
14. The composition of claim 1 , wherein each oligonucleotide of the plurality is independently Teo *R m5Ceo *R Aeo *R m5Ceo *R Teo *R Teo *R Teo *S m5Ceo *S Aeo *S Teo *S Aeo *S Aeo *R Teo *R Geo *R m5Ceo *R Teo *R Geo *R Geo (SEQ ID NO: 215) or a pharmaceutically acceptable salt thereof,
wherein each *R independently represents a phosphorothioate internucleotidic linkage in the Rp configuration, each *S independently represents a phosphorothioate internucleotidic linkage in the Sp configuration, each m5Ceo independently represents a 5-methyl 2′-O-methoxyethyl C, and each eo independently represents a 2′-O-(2-methoxyethyl) modification.