Asymmetric Interfering RNA Composition for Reduced Off-Target Effects
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Solution Overview
Problem
Current RNAi-based therapies face challenges such as limited efficacy, non-specific effects like interferon-like responses, and high costs associated with siRNA synthesis, as well as off-target gene silencing and cellular immune responses.
Innovation Solution
The development of asymmetrical interfering RNAs (aiRNAs) with a novel structural design featuring length asymmetry between the two RNA strands, allowing for a shorter RNA duplex structure that reduces interferon-like responses and synthesis costs, while enhancing efficacy, potency, and durability of gene silencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If symmetric siRNA structure (19-21 nucleotides with 3' overhangs) is used, then gene silencing can be achieved, but interferon-like responses are triggered and synthesis costs are high
Solution Approach 1:
The patent applies asymmetry by using asymmetric interfering RNAs (aiRNAs) with unequal strand lengths (e.g., 21 nt and 15 nt strands) instead of the conventional symmetric siRNA structure. This structural asymmetry reduces recognition by Toll-like receptors and other pattern recognition receptors that trigger interferon-like responses, while maintaining effective gene silencing through the asymmetric RISC complex formation and target mRNA cleavage mechanism.
2Reliability
If symmetric siRNA structure (19-21 nucleotides) is used, then gene silencing can be achieved, but synthesis costs are prohibitive
Solution Approach 1:
The asymmetric interfering RNA structure allows for reduced synthesis costs compared to conventional symmetric siRNAs. The shorter overall length and asymmetric structure enable more efficient chemical synthesis processes, reducing the number of synthesis cycles and reagent requirements while maintaining effective gene silencing activity.
Solution Approach 2:
The patent changes the structural parameters of the interfering RNA by using asymmetric strand lengths (e.g., 21 nt and 15 nt) instead of the conventional symmetric 19-21 nt structure. This parameter change optimizes the balance between gene silencing efficacy and synthesis cost, as the asymmetric structure requires fewer nucleotides to be synthesized while maintaining functional activity through enhanced RISC complex formation and target mRNA cleavage.
3Reliability
If conventional siRNA is used, then gene silencing can be achieved, but off-target gene silencing occurs
Solution Approach 1:
The asymmetric interfering RNA structure reduces off-target effects by creating a more specific interaction profile with the RISC complex and target mRNAs. The asymmetric strand lengths result in distinct binding characteristics that enhance specificity for the intended target while minimizing non-specific interactions, thereby reducing off-target gene silencing.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
aiRNAs demonstrate improved efficacy, potency, rapid onset, and durability in inducing gene silencing compared to traditional siRNAs, with reduced off-target effects and interferon-like responses, making them more suitable for therapeutic applications.
Implementation Method 1
Gene silencing through RNAi (RNA-interference) by use of small or short interfering RNA (siRNA) has emerged as a powerful tool for molecular biology
Implementation Method 2
genetic elements encoding short-hairpin RNAs (shRNAs) that are subsequently cleaved into siRNAs by the ribonuclease III-like enzyme, Dicer
Implementation Method 3
RISC binds, unwinds, and incorporates the anti-sense siRNA strand, which then recognizes and targets perfectly complementary mRNAs for cleavage
Data Source
AI summary
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.