Compounds and methods for enhanced cellular uptake
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Publication Number
US-9506030-B2
Publication Date
2016-11-29
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 disclosed invention relates to conjugated modified oligonucleotide compounds in which a modified oligonucleotide is linked through a scaffold or linker to a conjugate or ligand system. The compounds define scaffold-linker components and internucleoside linkages selected as phosphodiester or phosphorothioate, with each nucleoside at positions N_m or N^m independently modified or unmodified and m from 1 to 5.
The disclosure expands the scope of modified oligonucleotide structures by specifying sugar and internucleoside modification options, including beta-D-ribose, beta-D-deoxyribose, 2'-O-methoxy, 2'-O-methyl, 2'-fluoro, ribose, deoxyribose, 2'-modified nucleosides, and bicyclic sugar moieties such as cEt, LNA, ENA, and BNAs. It also describes fully modified and uniformly modified variants, gapmer motifs, microRNA mimic embodiments, and additional conjugation moieties linked to the scaffold or linker system.
The linkage and ligand system is described as enabling lectin-mediated uptake via C-type lectins, with emphasis on ASGPR using galactose or N-acetylgalactosamine ligands. The invention is applied to target RNA including microRNA, messenger RNA, pre-messenger RNA, and long noncoding RNA, and the disclosure also describes hybridization of the modified oligonucleotide to a complementary second modified oligonucleotide.
Claims Coverage
The consolidated claim coverage includes two independent claims: one directed to a conjugated modified oligonucleotide compound and one directed to a process of making the same type of conjugated modified oligonucleotide compound. The inventive features center on the defined N_m or N^m nucleoside/linkage architecture, phosphodiester or phosphorothioate internucleoside linkages, solid-support formation of the modified oligonucleotide, DMTr deprotection, sequential phosphoramidite coupling, and release of the conjugated modified oligonucleotide.
Conjugated modified oligonucleotide with defined N_m or N^m linkage architecture
A compound in which each N_m or N^m is independently a modified or unmodified nucleoside with m from 1 to 5, where X1 and X2 or X^1 and X^2 are each independently a phosphodiester linkage or a phosphorothioate linkage, and when m is greater than 1 each modified or unmodified nucleoside is connected to adjacent nucleosides by a phosphodiester internucleoside linkage or a phosphorothioate internucleoside linkage.
Solid-support synthesis with DMTr deprotection and sequential phosphoramidite coupling
A process comprising providing a solid support containing a conjugate, deprotecting the DMTr group under conditions effective to produce a reactive hydroxyl, performing sequential phosphoramidite coupling steps to form N_m or N^m, performing sequential phosphoramidite coupling steps to form the modified oligonucleotide, and releasing the conjugated modified oligonucleotide from the solid support.
The independent claims are directed to the defined conjugated modified oligonucleotide architecture and a solid-support process for building and releasing that conjugated modified oligonucleotide, with recurring structural limits of N_m or N^m and phosphodiester or phosphorothioate linkages. Dependent claims further narrow the structure and RNA targeting relationships, including complementarity, identity, and hybridization features.
Stated Advantages
Improved potency of conjugated modified oligonucleotides compared to unconjugated modified oligonucleotides.
Improved in vivo potency for GalNAc-conjugated modified oligonucleotides, including reported ED50/ED90 improvements.
Receptor-mediated uptake.
Lectin-mediated uptake via C-type lectins, including ASGPR using galactose or N-acetylgalactosamine ligands, is described.
Release or metabolism to unconjugated oligonucleotides is described, supporting functional activity in vivo.
Differential tissue distribution is described, including localization in liver versus kidney.
Onset and duration are described as extending over weeks.
In vivo potency and liver concentration outcomes are described.
Documented Applications
Target RNA applications including microRNA, messenger RNA, pre-messenger RNA, and long noncoding RNA.
In vivo use cases described for GalNAc-conjugated anti-miR-122 and anti-miR-21, including potency and liver concentrations.
GalNAc-conjugated modified oligonucleotides assessed in an ALDOA derepression context for miR-122, including quantitative outcomes such as ED50/ED90 improvements.
PTEN mRNA reduction using RNaseH gapmers.
miR-21 target contexts in tumor models, including reported tumor model outcomes.
Cholesterol conjugates described as improving potency, including cholesterol conjugation used in the documented efficacy contexts.
Therapeutic-use rationales connected to target RNA diseases and microRNA targeting in liver, hepatocyte, macrophage, and dendritic cell contexts.
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