Compounds and methods for enhanced cellular uptake
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Publication Number
US-10240151-B2
Publication Date
2019-03-26
Expiration Date
Abstract
Described herein are conjugated modified oligonucleotides that are complementary to a target RNA. The conjugate facilitates cellular uptake of the modified oligonucleotide, resulting improved potency.
Core Innovation
The invention relates to a compound comprising a linked nucleoside structure and a modified oligonucleotide (MO). Each N of N_m is independently a modified or unmodified nucleoside, where m is 1, 2, or 3, and at least one of X1 and X2 is a phosphodiester linkage. The MO consists of 8 to 25 linked nucleosides connected by phosphodiester internucleoside linkages or by phosphorothioate internucleoside linkages.
The core structural framework defines internucleoside linkage identity using O versus S linkages and includes linker or conjugation moiety features and alkyl substituents. When m is greater than 1, adjacent nucleosides are connected by phosphodiester or phosphorothioate internucleoside linkages, and the document also characterizes phosphodiester and phosphorothioate motif options for the MO.
The document further describes carbohydrate-conjugated compounds that interact with C-type lectins, including ASGPR with galactose or GalNAc, to enhance cellular uptake in cell types including Kuppfer cells, macrophages, endothelial cells, monocytes, leukocytes, dendritic cells, B cells, and hepatocytes. It also frames target RNA engagement by describing modified oligonucleotide complementarity to target RNA, including microRNAs and anti-miR targeting, and describes representative MO variants including gapmer and fully or uniformly modified oligonucleotides.
The document additionally specifies example sugar moiety options that can be used in the MO, including β-ribose/β-deoxyribose and modified sugars such as 2'-O-methyl, 2'-O-methoxyethyl, 2'-fluoro, and bicyclic sugars such as cEt, LNA, and ENA. The claimed and described ranges include oligonucleotide lengths of 7–25 or 8–30 in the document context.
Claims Coverage
The claim set is anchored by a single independent compound claim defining a linked nucleoside/modified oligonucleotide structure with specified phosphodiester/phosphorothioate linkage requirements and an 8 to 25 linked nucleoside MO length. Dependent claims refine the compound by constraining sequence relationships, hybridization relationships, and selectable sugar and linkage options, adding multiple inventive feature boundaries around the independent structure.
Linked nucleoside structure with modified or unmodified nucleosides and phosphodiester requirement
Each N of N_m is independently a modified or unmodified nucleoside with m being 1, 2, or 3, and at least one of X1 and X2 is a phosphodiester linkage.
Modified oligonucleotide length and linkage types
MO is a modified oligonucleotide consisting of 8 to 25 linked nucleosides connected by phosphodiester internucleoside linkages or by phosphorothioate internucleoside linkages.
Adjacent nucleoside connection by phosphodiester or phosphorothioate when m is greater than 1
When m is greater than 1, each modified or unmodified nucleoside of N_m is connected to adjacent modified or unmodified nucleosides of N_m by a phosphodiester internucleoside linkage or a phosphorothioate internucleoside linkage.
MicroRNA nucleobase sequence identity constraint
The modified oligonucleotide has a nucleobase sequence that is at least 90% identical to the nucleobase sequence of a microRNA.
Hybridization with a complementary second modified oligonucleotide
A modified oligonucleotide is hybridized with a second modified oligonucleotide whose nucleobase sequence is complementary to the first modified oligonucleotide’s nucleobase sequence.
Selectable sugar moiety set including 2′-O-methyl, 2′-O-methoxyethyl, 2′-fluoro, or bicyclic sugars
Each modified sugar moiety is independently chosen from 2′-O-methyl, 2′-O-methoxyethyl, 2′-fluoro, or bicyclic sugar moieties.
Specific nucleoside selection for N3
N3 is either β-D-deoxyriboadenosine or β-D-deoxyriboguanosine.
Phosphodiester-only internucleoside linkage for N_m connections
Each N of N_m is connected via a phosphodiester internucleoside linkage.
Overall, the claim coverage centers on a compound defined by a linked nucleoside structure with modified or unmodified nucleosides and explicit phosphodiester or phosphorothioate internucleoside linkage rules, combined with an MO comprising 8 to 25 linked nucleosides. Dependent claims further narrow the nucleobase sequence relative to microRNAs, hybridization and complementarity relationships, selectable sugar moiety options, and additional linkage and nucleoside identity constraints.
Stated Advantages
Potency improvements are reported, including ED50 and ED90 shifts.
Improved tissue distribution and release of unconjugated oligonucleotide are reported.
Duration of action is reported via ALDOA derepression and PTEN mRNA knockdown assays.
Enhanced cellular uptake.
Documented Applications
Anti-miR compounds directed to miR-122, including GalNAc- and cholesterol-conjugated examples.
Anti-miR-21 compounds and anti-miR-21 target context, including RNaseH gapmer constructs and PTEN mRNA knockdown.
RNaseH gapmer evaluation via ALDOA derepression and PTEN mRNA knockdown assays.
Tumor model readouts, including AFP biomarker measurements and target gene derepression.
Target RNA complementarity applications including microRNA targeting and anti-miR targeting, including human target RNA.
Cellular uptake enhancement using carbohydrate ligands such as galactose derivatives and N-acetylgalactosamine targeting lectins or ASGPR for uptake in cells such as hepatocytes.
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