Minor groove binder (MGB)-oligonucleotide miRNA antagonists
Inventors
Khvorova, Anastasia • Vermeulen, Annaleen • Kaiser, Rob • Karpilow, Jon • Vermeulen, Nicolaas M. J. • Mahoney, Walt
Assignees
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Publication Number
US-9334495-B2
Publication Date
2016-05-10
Expiration Date
Abstract
Compositions and methods for inhibiting the actions of non-coding RNAs such as miRNAs and piRNAs are provided. The compositions comprise single or double stranded oligonucleotides conjugated with Minor Groove Binders (“MGBs”). The oligonucleotides can vary in length, can contain nucleotides having one or more modifications, and have regions that are substantially complementary to one or more mature miRNAs or piRNAs.
Core Innovation
The invention provides an inhibitor composition for inhibiting non-coding RNAs. The composition comprises an oligonucleotide and a minor groove binder (MGB), and the oligonucleotide comprises nucleotides whose ribofuranose has Rz as —OCH3 and are linked by natural phosphodiester linkages. The MGB is conjugated to the 5′ end of the oligonucleotide.
The oligonucleotide is directed to a mature miRNA and/or piRNA target and can include regions substantially complementary to a mature miRNA/piRNA. The oligonucleotide may be a single- or double-stranded oligonucleotide and may include optional secondary structures.
The invention also discloses specific MGBs, including DPI3 and CDPI3, and linker/conjugation positioning options for attaching the MGB to the oligonucleotide. The document further describes inhibiting RNA activity using the inhibitor compositions and assessing inhibition using reporter assays, including a psiCHECK-2 dual luciferase assay with miRNA target sites.
Claims Coverage
The partial claim set includes one independent claim. The independent claim centers on a composition that combines an oligonucleotide with a defined ribofuranose feature and natural phosphodiester linkages together with an MGB conjugated to the 5′ end, aimed at inhibiting non-coding RNAs; dependent claims refine targeting, MGB identity/structure, and additional oligonucleotide details.
MGB-conjugated 5′-oligonucleotide inhibitor for non-coding RNAs
An inhibitor composition for inhibiting non-coding RNAs comprising an oligonucleotide and a minor groove binder (MGB), wherein the oligonucleotide comprises nucleotides having ribofuranose with Rz as —OCH3 and B as a normal base or a modified base, wherein the nucleotides are linked by natural phosphodiester linkages, and wherein the MGB is conjugated to the 5′ end of the oligonucleotide.
Complementary mature target strand oligonucleotide
The inhibitor composition wherein the oligonucleotide contains a sequence complementary to a mature strand of a target sequence.
DPI3 or CDPI3 MGB
The inhibitor composition specifying that the MGB is DPI3 or CDPI3.
Specified modified bases for the oligonucleotide
The inhibitor composition wherein the oligonucleotide includes one or more specified modified bases selected from a defined list of modified nucleosides/nucleobases and related substituted base analogs.
MGB structural selection with linker/substituent constraints
The inhibitor composition further defined by the MGB having a structure selected from provided chemical structures, where m is an integer from 2 to 5, r is an integer from 2 to 10, and substituents Ra and Rb are each independently a linker to an oligonucleotide or a fluorophore with specified Rc and Rd substituent options.
Method inhibiting miRNA activity in vitro or in vivo
A method that inhibits miRNA activity in vitro or in vivo by introducing the inhibitor composition to a location where miRNA activity exists.
Overall, the claim coverage centers on a non-coding RNA inhibitor composition defined by a ribofuranose feature (Rz as —OCH3) and natural phosphodiester linkages, together with an MGB conjugated to the 5′ end, with dependent refinements specifying mature-target complementarity, particular MGB identities (e.g., DPI3/CDPI3) and MGB structural/linker parameters, and additional refinement to miRNA inhibition in vitro or in vivo.
Stated Advantages
Improved potency of MGB-2′-OMe constructs compared to 2′-O-methyl controls, with reported improvements such as about 3.4-fold for 5′-MGB-2′-OMe let-7c and about 10×/about 3× for miR-21 depending on MGB position.
Minimal observed toxicity.
Documented Applications
Therapeutic and prophylactic use cases including reversing therapeutic siRNA/miRNA/piRNA actions.
Diagnostic use cases.
In vitro or in vivo inhibition of miRNA activity by introducing the inhibitor composition to a location where miRNA activity exists.
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