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-9328345-B2
Publication Date
2016-05-03
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 is directed to asymmetric interfering RNA (aiRNA) as a non-siRNA RNAi scaffold. It comprises an antisense strand and a sense strand in an asymmetric RNAi duplex that supports target RNA reduction, with a defined antisense strand length and independent 3′ and 5′ overhangs and a shorter sense strand forming a defined double-stranded region.
The antisense strand is described as complementary to a target RNA at the 3′ overhang and the double-stranded region, and the 3′ overhang of the antisense strand consists of RNA nucleotides. The document further states that the duplex has a defined base-pair region and may include GC content constraints.
The aiRNA framework is associated with RNA interference, including selective target mRNA cleavage through the RNA-induced silencing complex (RISC) and Argonaute 2 (Ago2). Compared with siRNA, the aiRNA is described as entering RISC more efficiently and preferentially loading the antisense strand while avoiding interferon-response gene induction and sense-strand off-target silencing.
The document also describes optional nucleotide-level features for the asymmetric interfering RNA duplex, including instability-reducing stabilization and optional chemical modifications of the duplex components, such as sugar/backbone/base modifications, phosphothioate, non-natural bases, deoxynucleotides, 2′-O substitutions, and conjugation and delivery.
Claims Coverage
The independent claim coverage centers on a structurally defined asymmetric interfering RNA duplex with three core inventive features: a defined antisense strand with terminal overhangs, a complementary sense strand that forms a defined base-pair region, and optional sequence and nucleotide/analog refinements including GC content and modifications. The claim set also includes therapeutic use cases.
Asymmetric interfering RNA duplex with defined antisense length and terminal overhangs
An asymmetric interfering RNA duplex molecule comprising an antisense strand consisting of 19-23 nucleotides, wherein the antisense strand includes a 2-4 nucleotide 3′ overhang and a 2-4 nucleotide 5′ overhang, wherein the antisense strand at the 3′ overhang and the double-stranded region is complementary to a target RNA, and wherein the 3′ overhang of the antisense strand consists of RNA nucleotides.
Complementary sense strand forming a defined base-pair double-stranded region
A sense strand consisting of 14-17 nucleotides, wherein the sense strand is complementary to the antisense strand and forms a 14, 15, 16, or 17 base pair double-stranded region with the antisense strand.
GC content constraint for the double-stranded region
The double-stranded region has a GC content between 30% and 70%.
2′ sugar substitution options for modified nucleotides or analogues
A modified nucleotide or analogue includes a sugar-modified ribonucleotide whose 2′-OH group is replaced by H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, with each R independently C1-C6 alkyl, C1-C6 alkenyl, or C1-C6 alkynyl, and each halo being F, Cl, Br, or I.
Phosphothioate backbone modification
A backbone-modified ribonucleotide containing a phosphothioate group.
Treating a disease or condition by administering the RNA duplex
A method of treating a disease or condition, comprising administering to a subject in need the RNA duplex molecule described in claim 1.
Treating an ocular disease by administering the RNA duplex
A method of treating an ocular disease, comprising administering to a subject in need thereof the RNA duplex molecule described in claim 1.
Overall, the claim coverage centers on an asymmetric interfering RNA duplex defined by an antisense strand with independent 3′ and 5′ overhangs and a sense strand that forms a defined base-pair double-stranded region, with further refinements for GC content and optional sugar and backbone modifications, and with therapeutic use cases including disease treatment and ocular-disease treatment.
Stated Advantages
Enters RISC more efficiently than siRNA.
Selectively cleaves target mRNA.
Avoids interferon-like responses and sense-strand off-target silencing.
Shows stronger potency, rapidity, and durability in cell and xenograft tumor models.
Demonstrates sequence/allele specificity in described models.
Provides potent, gene-specific, rapid-onset, and durable silencing compared with conventional siRNA.
Reduces target mRNA levels within about 24 h.
Induces sequence-specific cleavage fragments within the antisense target region.
Efficiently loaded into Ago/RISC with preferential antisense-strand retention.
Shows allele-specific silencing.
Does not elicit interferon-response gene induction compared with siRNA.
Abolishes non-specific sense-strand mediated silencing.
In vivo, produces stronger xenograft tumor growth inhibition than PEI-complexed β-catenin siRNA without significant body weight change.
Documented Applications
Treating a disease or condition by administering the RNA duplex molecule to a subject in need.
Treating an ocular disease by administering the RNA duplex molecule to a subject in need thereof.
In vivo colon cancer xenograft models using PEI-complexed β-catenin aiRNA for tumor growth inhibition.
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