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-10927376-B2
Publication Date
2021-02-23
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 disclosure provides an asymmetric interfering RNA (aiRNA) duplex molecule including an antisense strand and a sense strand, where the antisense strand is longer than the sense strand. The antisense strand includes a 3′-overhang and, when duplexed with the sense strand, forms a 5′-blunt end. The sense strand forms a double-stranded region with the antisense strand, with defined matching base-pair and mismatch-pair options.
The antisense strand has a sequence that specifically targets an expressed nucleotide sequence of a target gene through a perfectly complementary section with the target sequence. The antisense strand optionally further comprises a single-nucleotide or dinucleotide non-complementary section between the 5′ end of the antisense strand and the target sequence, where the non-complementary section is selected from A, U, T.
The disclosure further supports aiRNA duplexes for gene silencing in eukaryotic cells that provide sequence-specific mRNA cleavage and improved pathway engagement. Documented results describe improved RISC/Ago2 loading versus siRNA and the reduction or abolition of sense-strand-mediated off-target silencing, and additional described outcomes include minimized interferon-like responses and anti-tumor activity in xenograft mouse models using an aiRNA targeting β-catenin.
Claims Coverage
The provided independent claim defines the aiRNA duplex with length asymmetry, specific terminal structure, and a target-gene antisense sequence designed for specific pairing, comprising multiple ranges of matching and mismatch base pairs plus an optional non-complementary section. The inventive coverage is focused on three core aspects: antisense/sense length and terminal architecture, defined duplex region pairing/mismatch structure, and sequence complementarity to an expressed target nucleotide sequence with an optional internal non-complementary A/U/T segment.
Asymmetric duplex with longer antisense and defined terminals
An asymmetric interfering RNA duplex molecule comprising an antisense strand and a sense strand, where the antisense strand is longer than the sense strand, consists of 19, 20, 21, 22 or 23 nucleotides and includes a 3′-overhang of 2-9 nucleotides as part of the 19-23 nucleotides’ length, and has a 5′-blunt end when duplexed with the sense strand.
Defined double-stranded region with matching and mismatch pair options
The sense strand consists of 12, 13, 14, 15, 16 or 17 nucleotides, forms a double-stranded region with the antisense strand where the double-stranded region is selected from the group consisting of 10-15 matching base pairs plus 2 mismatch pairs, 11-16 matching base pairs plus 1 mismatch pair, and 12-17 perfectly matching base pairs.
Antisense targeting expressed nucleotide sequence with optional non-complementary A/U/T section
The antisense strand has a sequence that specifically targets an expressed nucleotide sequence of a target gene through a perfectly complementary section with the target sequence and optionally further comprises a single-nucleotide or dinucleotide non-complementary section between the 5′ end of the antisense strand and the target sequence, the non-complementary section consisting of nucleotide(s) selected from A, U and T.
Across the provided independent claim, protection is concentrated on an aiRNA duplex defined by antisense length being longer than the sense strand, specific terminal architecture, a defined duplex-region pairing/mismatch structure, and an antisense sequence that specifically targets an expressed target-gene nucleotide sequence via a perfectly complementary section with an optional A/U/T non-complementary single- or dinucleotide segment.
Stated Advantages
Improved RISC/Ago2 loading versus siRNA.
Abolition of sense-strand-mediated off-target silencing.
Minimized interferon-like responses.
Sequence-specific mRNA cleavage that is described as rapid, potent, and durable.
Demonstrated anti-tumor activity in xenograft mouse models targeting β-catenin.
Documented Applications
Gene silencing in eukaryotic cells using an asymmetric interfering RNA duplex targeting an expressed nucleotide sequence of a target gene, including mammalian and avian cells.
Anti-tumor activity in xenograft mouse models targeting β-catenin / Wnt signaling.
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