Segmented siRNA Passenger Strand for Reduced Off-Target Effects
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Solution Overview
Problem
Current siRNA therapies face challenges due to off-target effects and low specificity, largely attributed to the unintentional incorporation of passenger strands into RNA-induced silencing complexes, leading to unintended gene silencing and interferon responses, which complicates their therapeutic and gene discovery applications.
Innovation Solution
The development of RNA complexes with a discontinuous passenger strand and LNA nucleotide analogues, where the antisense strand is specifically designed to be incorporated into the RISC complex, reducing passenger strand loading and enhancing stability, thereby increasing targeting specificity and avoiding interferon induction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If continuous passenger strands are used in siRNA complexes, then the complexes can be synthesized and delivered, but off-target effects occur due to unintentional incorporation of passenger strands into RISC complexes
Solution Approach 1:
The passenger strand is divided into multiple short segments (e.g., two 10-mer segments) separated by a gap of 1-4 nucleotides. This segmentation prevents the passenger strand from being incorporated into the RISC complex as a continuous strand, thereby eliminating off-target effects while maintaining the ability to deliver the siRNA complex to cells.
Solution Approach 2:
The harmful continuous passenger strand is extracted and replaced with segmented passenger strands. By removing the continuity of the passenger strand, the invention extracts the problematic element (continuous passenger strand incorporation into RISC) while retaining the beneficial elements of the siRNA complex structure.
2Stability of the object's composition
If heavily modified antisense strands are used to enhance stability, then biostability improves, but the complexity of synthesis increases
Solution Approach 1:
Chemical modifications (such as 2'-O-methyl, LNA, or phosphorothioate modifications) are applied selectively at specific positions within the antisense strand rather than uniformly throughout. This local quality approach enhances biostability and nuclease resistance at critical regions while maintaining synthesis feasibility and RISC loading efficiency.
3Reliability
If standard siRNA complexes are used, then gene silencing can be achieved, but interferon responses are induced reducing therapeutic applicability
Solution Approach 1:
The invention changes key parameters of the siRNA complex structure: the passenger strand is segmented with gaps of 1-4 nucleotides, and the overall length is controlled (typically 19-25 nucleotides per segment). These parameter changes prevent interferon response induction while maintaining effective gene silencing efficacy through RISC-mediated antisense strand activity.
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
This approach significantly enhances the specificity of gene silencing, reduces off-target effects, and improves the stability and efficacy of siRNA complexes in vivo, allowing for the use of heavily modified antisense strands and minimizing interferon responses.
Implementation Method 1
the core double stranded region comprising an antisense strand and a discontinuous passenger strand that is hybridised to the antisense strand, wherein the RNA complex comprises LNA nucleotide analogues which increase the melting temperature of the core double stranded region
Data Source
Figure 1~1B
Figure 2
Figure 3
AI summary
The present invention is directed to pharmaceutical and therapeutic compositions which comprise RNA complexes comprising an antisense strand and a discontinued passenger strand capable of regulating gene expression. The use of a discontinued passenger strand reduces off target effects of the RNA complexes and also has other advantages.