Compositions of asymmetric interfering RNA and uses thereof
Inventors
Li, Chiang Jia • Sun, Xiangao • Rogoff, Harry • Li, Youzhi
Assignees
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Publication Number
US-10266821-B2
Publication Date
2019-04-23
Expiration Date
Abstract
The present invention provides asymmetrical duplex RNA molecules that are capable of effecting sequence-specific gene silencing. The RNA molecule comprises a first strand and a second strand. The first strand is longer than the second strand. The RNA molecule comprises a double-stranded region formed by the first strand and the second strand, and two ends independently selected from the group consisting of 5′-overhang, 3′-overhang, and blunt end. The RNA molecules of the present invention can be used as research tools and/or therapeutics.
Core Innovation
The invention relates to asymmetric interfering RNA (aiRNA) duplexes in which a sense oligonucleotide is longer than a complementary antisense oligonucleotide, forming a double-stranded region with strand-length asymmetry. The duplex includes independently selectable terminal ends, including 3′-overhangs and 5′-overhangs or blunt ends, and the antisense oligonucleotide has sequence characteristics and end features that support sequence-specific gene silencing in eukaryotic cells, including mammalian and avian cells.
The duplex is configured so that the antisense oligonucleotide has at least 70% complementarity to an expressed nucleotide sequence of a target gene. The sense and antisense oligonucleotides are paired such that 10–17 bases of the sense oligonucleotide are complementary with bases in the antisense oligonucleotide to form the double-stranded region, and at least the first base and last base of the sense oligonucleotide base pair with bases of the antisense oligonucleotide. The sense oligonucleotide is stated not to substantially mediate off-target silencing.
Mechanistic and functional disclosures support that aiRNA mediates sequence-specific mRNA cleavage in the RNA-induced silencing complex (RISC), including involvement of Dicer and Argonaute 2 (Ago2). The disclosures further state improvements relative to siRNA, including faster onset, greater potency/efficacy/durability, enhanced RISC loading and antisense strand retention, reduced sense-strand mediated off-target silencing, reduced interferon-like response associated with dsRNA-dependent protein kinase (PKR) and Toll-like receptors (TLR), and enhanced serum stability.
The invention further addresses duplex architecture options and chemical/formulation choices, including restrictions on terminal ribonucleotides, overhang features, and the ability to include modifications and additional components such as phosphorothioate and 2′-O-methyl, sugar-modified ribonucleotides, backbone-modified ribonucleotides, and tritylated base. The duplex and related compositions are presented for broad utility as research reagents, gene-expression modulation agents, pharmaceutical compositions, kits, vectors, and cells, along with described delivery and formulation approaches using pharmaceutically acceptable excipients/carriers.
Claims Coverage
The independently claimed subject matter is a method for preparing an asymmetric interfering RNA duplex with strand-length asymmetry and independently selectable terminal ends, incorporating sequence complementarity and an off-target silencing limitation. From the provided claim set, 1 independent claim is identified, and its main inventive features are the duplex architecture, specific overhang features and terminal nucleotide restriction, sufficient target complementarity, and the requirement that the sense oligonucleotide does not substantially mediate off-target silencing.
Asymmetric duplex strand lengths and double-stranded region pairing
A method for preparing an asymmetric interfering RNA duplex comprising combining a sense oligonucleotide consisting of 14, 15, 16 or 17 nucleotides and an antisense oligonucleotide consisting of 19, 20, 21, 22 or 23 nucleotides, wherein 10, 11, 12, 13, 14, 15, 16, or 17 bases of the sense oligonucleotide are complementary with bases in the antisense oligonucleotide to form a double-stranded region, and at least the first base and last base of the sense oligonucleotide base pair with bases of the antisense oligonucleotide.
Independent terminal overhang features
The antisense oligonucleotide includes a 3′-overhang of 1, 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides and a 5′-overhang of 0, 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides.
High complementarity to an expressed target gene sequence with 3′ terminal ribonucleotide
The antisense oligonucleotide has a sequence that is at least 70% complementary to an expressed nucleotide sequence of a target gene and has a last nucleotide at its 3′ end consisting of an A, U, G or C ribonucleotide.
Avoidance of substantial sense-mediated off-target silencing
The sense oligonucleotide of the RNA duplex does not substantially mediate off-target silencing.
Across the provided independent claim, coverage centers on constructing an asymmetric interfering RNA duplex with longer sense and shorter antisense strands, a defined double-stranded region formed by specified complementary base pairing, antisense-defined terminal overhangs, at least 70% complementarity to an expressed target gene sequence with a restricted 3′ terminal ribonucleotide, and an explicit limitation that the sense oligonucleotide does not substantially mediate off-target silencing.
Stated Advantages
Faster onset than siRNA.
Greater potency/efficacy/durability than siRNA.
Enhanced RISC loading and antisense strand retention.
Reduced sense-strand mediated off-target silencing.
Reduced interferon-like response associated with PKR and Toll-like receptors (TLR).
Enhanced serum stability.
Demonstrated in vivo anti-tumor activity in xenograft mouse models.
Documented Applications
Sequence-specific gene silencing in eukaryotic cells, including mammalian/avian cells.
Use as a research reagent for gene-expression modulation.
Gene-expression modulation methods.
Pharmaceutical compositions.
Kits.
Expression vectors.
Cells.
Delivery and formulation approaches using pharmaceutically acceptable excipient/carrier.
In vivo anti-tumor activity in xenograft mouse models.
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