siRNA Sequence-Independent Modifications for Off-Target Reduction
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Solution Overview
Problem
Current RNA interference technologies face challenges in reducing sequence-independent off-target phenotypic effects, as increasing strand bias alone is not sufficient to minimize these effects, and existing methods fail to maintain potency while reducing off-target effects effectively.
Innovation Solution
The development of chemically synthesized short interfering RNAs (siRNAs) with specific sequence-independent modification formats, including passenger and guide oligonucleotides, that are stable to nucleases and reduce off-target events while maintaining potency, comprising modified nucleotides and structures that enhance strand bias and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If strand bias is increased to reduce off-target effects, then specificity is improved, but potency is reduced
Solution Approach 1:
The patent applies local quality by introducing modifications at specific positions within the siRNA sequence rather than uniformly across the entire molecule. Different modification types (2'-O-methyl, LNA, phosphorothioate) are placed at different positions to locally enhance strand bias while preserving overall potency. This localized approach allows precise control over which strand is incorporated into RISC without compromising the overall functionality of the siRNA.
Solution Approach 2:
The patent utilizes parameter changes by systematically varying modification types, positions, and combinations to optimize the balance between strand bias and potency. By changing chemical parameters (modification type), positional parameters (location within sequence), and compositional parameters (number of modifications), the patent achieves enhanced specificity while maintaining therapeutic effectiveness.
2Manufacturing precision
If modified nucleotides are introduced to enhance strand bias, then off-target effects are reduced, but molecular stability is compromised
Solution Approach 1:
The patent employs composite materials by combining multiple modification types within a single siRNA molecule. The duplex structure integrates modified nucleotides (2'-O-methyl, LNA) with unmodified nucleotides, and incorporates different chemical modifications at strategic positions. This composite approach creates a molecule that simultaneously achieves enhanced strand bias through modified nucleotides while maintaining molecular stability through the balanced incorporation of various modification types and the protective duplex structure.
3Reliability
If sequence-independent modifications are used to reduce off-target effects, then reliability is improved, but manufacturing complexity increases
Solution Approach 1:
The patent applies parameter changes by establishing systematic rules for modification placement that can be applied across different siRNA sequences. By defining specific modification types at defined positions relative to the guide strand (e.g., 2'-O-methyl at positions 1-3 from the 5' end), the patent creates a standardized approach that enhances reliability across various targets while providing a manufacturable framework that reduces the need for sequence-by-sequence optimization.
Data Source
AI summary
Modification formats having modified nucleotides are provided for siRNA. Short interfering RNA having modification formats and modified nucleotides provided herein reduce off-target effects in RNA interference of endogenous genes. Further modification formatted siRNAs are demonstrated to be stabilized to nuclease-rich environments. Unexpectedly, increasing or maintaining strand bias, while necessary to maintain potency for endogenous RNA interference, is not sufficient for reducing off-target effects in cell biology assays.