Oligonucleotide compositions and methods of use thereof
Inventors
Vargeese, Chandra • Iwamoto, Naoki • Butler, David Charles Donnell • Marappan, Subramanian • Lu, Genliang • Zhang, Jason Jingxin • VATHIPADIEKAL, Vinod • Frank-Kamenetsky, Maria David • Apponi, Luciano Henrique • Cho, Young Jin
Assignees
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Publication Number
US-11603532-B2
Publication Date
2023-03-14
Expiration Date
Abstract
Among other things, the present disclosure provides designed oligonucleotides, compositions, and methods thereof. In some embodiments, provided oligonucleotide compositions provide improved single-stranded RNA interference and/or RNase H-mediated knockdown. Among other things, the present disclosure encompasses the recognition that structural elements of oligonucleotides, such as base sequence, chemical modifications (e.g., modifications of sugar, base, and/or internucleotidic linkages) or patterns thereof, conjugation with additional chemical moieties, and/or stereochemistry [e.g., stereochemistry of backbone chiral centers (chiral internucleotidic linkages)], and/or patterns thereof, can have significant impact on oligonucleotide properties and activities, e.g., RNA interference (RNAi) activity, stability, delivery, etc. In some embodiments, the present disclosure provides methods for treatment of diseases using provided oligonucleotide compositions, for example, in RNA interference and/or RNase H-mediated knockdown.
Core Innovation
The invention relates to an oligonucleotide composition comprising a plurality of oligonucleotides that are structurally identical, wherein the level of the plurality in the composition is predetermined. The oligonucleotides are about 10 to about 50 nucleotides in length and include at least 5 internucleotidic linkages that are chirally controlled. Each oligonucleotide includes a 5′-end nucleoside or a salt form thereof, and/or a 5′-end sugar or a salt form thereof.
The disclosed subject matter further defines chirally controlled phosphorothioate internucleotidic linkages, including embodiments in which the phosphorothioate internucleotidic linkage is in the Sp configuration. Dependent refinements also include quantitative constraints on the proportion of oligonucleotides having a specified number of chirally controlled internucleotidic linkages, and a threshold in which each oligonucleotide has at least 50% of its nucleotidic units containing a 2′-substitution.
The disclosure further includes a target-binding sequence that is completely complementary to a target sequence, with a length of at least 10–20 bases, where the target sequence contains one or more allelic sites that are SNPs or mutations. Across the provided material, the compositions are also described in RNAi, ssRNAi, and antisense oligonucleotide contexts, including RNase H-mediated knockdown and RNase H-dependent antisense oligonucleotide activity.
Claims Coverage
The independent claim coverage centers on an oligonucleotide composition comprising structurally identical oligonucleotides of about 10 to about 50 nucleotides, with a predetermined level in the composition, at least 5 chirally controlled internucleotidic linkages, and specified 5′-end nucleoside and/or 5′-end sugar features in nucleoside/sugar form or salt form. Dependent claims further constrain chirality, phosphorothioate stereochemistry, 2′-substitution, and target-binding sequence complementarity with allele-site context.
Structurally identical oligonucleotides with predetermined level
A plurality of oligonucleotides that are structurally identical, wherein the level of the plurality in the composition is predetermined.
About 10 to about 50 nucleotides in length
The oligonucleotides of the plurality are about 10 to about 50 nucleotides in length.
At least 5 chirally controlled internucleotidic linkages
At least 5 internucleotidic linkages are chirally controlled.
5′-end nucleoside or 5′-end sugar in salt form
The 5′-end nucleoside of each oligonucleotide is or a salt form thereof, or the 5′-end sugar of each oligonucleotide is or a salt form thereof.
Chirally controlled phosphorothioate internucleotidic linkages
Each oligonucleotide independently contains at least 5 chirally controlled phosphorothioate internucleotidic linkages.
Phosphorothioate internucleotidic linkage in the Sp configuration
The phosphorothioate internucleotidic linkage is a chirally controlled internucleotidic linkage in the Sp configuration.
At least 50% of nucleotidic units containing a 2′-substitution
Each oligonucleotide has at least 50% of its nucleotidic units containing a 2′-substitution.
Completely complementary target-binding sequence with SNP or mutation allele sites
A target-binding sequence that is completely complementary to a target sequence, with a length of at least 10–20 bases, where the target sequence contains one or more allelic sites that are SNPs or mutations.
Overall, the claims are directed to a structurally identical oligonucleotide composition of predetermined level and length, with at least 5 chirally controlled internucleotidic linkages and defined 5′-end nucleoside and/or 5′-end sugar features. The dependent features further specify chirally controlled phosphorothioate linkages in the Sp configuration, 2′-substitution thresholds, a composition-level chirality distribution constraint, and completely complementary target-binding sequences with SNP/mutation allele-site context.
Stated Advantages
No induction of a panel of cytokines/chemokines (IL-1β, IL-6, MCP-1, IL-12p40, IL-12p70, IL-1α, MIP-1α, MIP-1β, and TNFα) in any donor sample for the evaluated PNPLA3 ssRNAi agents.
Improved single-stranded RNAi activity.
Improved tissue delivery, including delivery to muscle tissue.
Improved stability in plasma and tissue homogenates.
The described oligonucleotides are reported to maintain or improve RNA interference activity despite absence of 5′ phosphates.
Improving in vivo duration and potency for stereopure versus stereorandom reference compositions for APOC3 ASOs.
Substantially greater knockdown at later time points for stereopure relative to stereorandom reference compositions.
Stronger liver mRNA and protein knockdown for stereopure relative to stereorandom reference compositions.
Up to greater liver exposure for stereopure relative to stereorandom reference compositions, with PK/PD correlation.
Improving treatment time/temperature.
Reducing desulfurization and support cleavage.
Improved knockdown versus stereorandom mixtures.
Increased stability against nucleases.
Preservation of RNAi activity while increasing nuclease stability.
Lipid conjugation can improve activity and distribution.
Lipid conjugation can reduce hTLR9 agonist activity.
Lipid conjugation potentially increases hTLR9 antagonist activity.
Reducing 2′-F can increase in vitro potency/stability while reducing toxicity.
Documented Applications
In vitro evaluation of PNPLA3 single-stranded RNAi (ssRNAi) oligonucleotides in Huh7 cells, with residual PNPLA3 mRNA levels normalized to HPRT1 and residual PNPLA3 mRNA reported across multiple conditions.
Cytokine-release assessment of PNPLA3 ssRNAi agents using donor samples, measuring induction of IL-1β, IL-6, MCP-1, IL-12p40, IL-12p70, IL-1α, MIP-1α, MIP-1β, and TNFα.
RNA interference (RNAi) and RNase H-mediated knockdown agents using the described oligonucleotide sugars/linkages and base sequences.
Ago-2 assays using RNA test substrate (WV-2372) are reported to show RNA interference and/or RNase H-mediated knockdown for named oligonucleotides, including those associated with APOC3 mRNA.
Tissue delivery to muscle tissue, including skeletal muscle.
Treating diseases using an RNAi-directed oligonucleotide composition, including embodiments related to RNase H-mediated knockdown and target genes ACVR2B, APOB, APOC3, FXI, KRT14, MSTN, PCSK9, and PNPLA3.
Therapeutic use of chirally controlled single-stranded RNAi agent and antisense oligonucleotide compositions for gene knockdown, including RNA interference (RISC), RNase H-mediated knockdown, steric hindrance of translation, and mRNA splicing/exon inclusion exclusion.
Co-administration with cholesterol modulating agents, including lipase inhibitor, HMG-CoA reductase inhibitor, CETP inhibitor, bile acid sequestrant, and PCSK9 modulator.
Treatment and knockdown of APOC3/hApo-III using GalNAc-conjugated phosphorothioate ASOs, including evaluation in human APOC3 transgenic mice contexts.
A stereocontrolled single-stranded RNAi platform for RNA interference knockdown, including analysis of nuclease cleavage sites.
Modulation of hTLR9 activity, including reducing hTLR9 agonist activity while potentially increasing hTLR9 antagonist activity.
Target-binding sequences and allelic-site discrimination in the context of disease-associated alleles, including use contexts aligned to RNA interference (RNAi), single-stranded RNAi (ssRNAi), and RNase H-dependent antisense oligonucleotide activity.
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