Modified dsRNA Agents with Seed Destabilization for Off-Target Reduction
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Solution Overview
Problem
Existing RNAi duplex agents, such as siRNAs, suffer from significant off-target effects due to miRNA-like interactions, which can lead to unintended gene silencing and potential toxicity, compromising their therapeutic efficacy.
Innovation Solution
Incorporation of thermally destabilizing modifications in the seed region of the antisense strand of dsRNA molecules, specifically within positions 2-9 of the 5′-end, to maintain on-target gene silencing efficacy while reducing off-target effects, achieved by adjusting the melting temperature to a range of 40° C. to 80° C. and employing chemical modifications like 2′-fluoro and phosphorothioate linkages.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If siRNA is used to induce RNA interference, then gene silencing efficacy is achieved, but off-target effects occur due to miRNA-like interactions
Solution Approach 1:
The patent applies local quality by introducing thermally destabilizing modifications specifically at positions 2-9 of the antisense strand (the seed region), while leaving other regions unchanged. This localized modification reduces off-target effects without compromising overall gene silencing efficacy, as the destabilizing effect is confined to the seed region where miRNA-like interactions occur.
Solution Approach 2:
The patent changes the thermal parameters of the siRNA duplex by incorporating modifications such as 2′-O-methyl, 2′-fluoro, or locked nucleic acid residues at positions 2-9 of the antisense strand. These parameter changes reduce the stability of seed region pairing, thereby reducing off-target effects while maintaining the overall melting temperature within the therapeutic range of 40-80°C.
2Object-generated harmful factors
If thermally destabilizing modifications are introduced in the seed region, then off-target effects are reduced, but gene silencing efficacy may be compromised
Solution Approach 1:
The patent carefully controls the melting temperature parameter by selecting modifications that destabilize the seed region but maintain the overall duplex Tm within 40-80°C. This parameter optimization ensures that off-target effects are reduced while gene silencing efficacy is preserved.
Solution Approach 2:
The patent uses composite chemical modifications, combining thermally destabilizing residues (such as 2′-O-methyl, 2′-fluoro, or locked nucleic acid) at positions 2-9 with standard nucleotides or stabilizing modifications at other positions. This composite approach allows the seed region to have reduced stability for minimizing off-target effects while other regions maintain sufficient stability for effective gene silencing.
3Reliability
If chemical modifications like 2′-fluoro and phosphorothioate linkages are employed, then therapeutic potency is maintained, but manufacturing complexity increases
Solution Approach 1:
The patent applies chemical modifications locally at specific positions (2′-fluoro at positions 2-9 and phosphorothioate linkages at selected positions) rather than throughout the entire siRNA sequence. This localized approach maintains therapeutic potency while reducing manufacturing complexity compared to full modification.
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
The modified dsRNA molecules demonstrate enhanced on-target gene silencing with reduced off-target gene silencing and toxicity, maintaining therapeutic potency and minimizing unintended gene regulation.
Implementation Method 1
the dsRNA molecule has a melting temperature in the range of from about 40° C. to about 80° C.
Data Source
AI summary
One aspect of the present invention relates to double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene. The antisense strand of the dsRNA molecule comprises at least one thermally destabilizing nucleotide occurring at a seed region; the dsRNA comprises at least four 2′-fluoro modifications, and the sense strand of the dsRNA molecule comprises ligand, wherein the ligand is an ASGPR ligand. Other aspects of the invention relate to pharmaceutical compositions comprising these dsRNA molecules suitable for therapeutic use, and methods of inhibiting the expression of a target gene by administering these dsRNA molecules, e.g., for the treatment of various disease conditions.