Programmable Cond-siRNA for Conditional Gene Silencing
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Solution Overview
Problem
Current RNAi therapy faces challenges in delivering RNAi agents specifically to disease-related cells due to poorly suppressed background drug activity, weak ON state drug potency, input and output sequence overlap, and short device lifetimes, limiting the effectiveness of conditionally activated oligonucleotide therapeutics in mammalian cells.
Innovation Solution
Development of a programmable, conditionally activated small interfering RNA (Cond-siRNA) construct comprising a sensor strand, a core strand, and a guide strand, which forms a single structure via complementary binding, allowing for specific activation by cellular RNA transcripts and improved stability and potency through chemical modifications such as LNA and 2'-O-methyl modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If strand displacement switches are used to create conditionally activated RNAi agents, then conditional activation capability is achieved, but background drug activity is poorly suppressed
Solution Approach 1:
The RNAi agent is divided into three separate strands: a guide strand, a core strand, and a sensor strand. The guide strand and core strand form an inactive RNAi duplex that is segmented from the active state by the sensor strand's blocking sequence. This segmentation allows the drug to remain inactive until the sensor strand is displaced by the target RNA, thereby suppressing background activity while maintaining conditional activation capability.
2Adaptability or versatility
If strand displacement switches are used for conditional activation, then programmability is achieved, but ON state drug potency is weak
Solution Approach 1:
The guide strand and core strand are pre-assembled into an RNAi duplex structure that is primed and ready for activity. The sensor strand is pre-positioned to block this structure. When the sensor strand is displaced by the target RNA, the pre-formed RNAi duplex is immediately activated without requiring additional assembly steps, thereby achieving strong ON state potency while maintaining programmability through the sensor strand sequence.
3Device complexity
If sensor duplex and RNAi duplex are attached via core strand, then single construct simplicity is achieved, but input and output sequence overlap occurs
Solution Approach 1:
The core strand is designed with differentiated regions: a first region that binds to the sensor strand to form the sensor duplex, and a second region that binds to the guide strand to form the RNAi duplex. This local differentiation ensures that the sequences in these regions are distinct and do not overlap with the input or output sequences, thereby preventing sequence overlap while maintaining the simplicity of a single construct design.
4Reliability
If conventional RNAi agents are used, then gene silencing capability is achieved, but device lifetime is short
Solution Approach 1:
The RNAi agent incorporates chemically modified nucleotides including 2'-O-methyl modifications and locked nucleic acid (LNA) modifications. These composite chemical structures enhance the stability and nuclease resistance of the RNA strands, thereby extending the device lifetime in biological environments while preserving the gene silencing capability through maintained structural integrity and function.
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 Cond-siRNA achieves significant silencing of target genes with over 90% efficiency in cells expressing sequence-matched RNA transcripts while strongly suppressing background RNAi activity in mismatched inputs, maintaining integrity over days in mammalian cytosol and enabling precise gene expression control.
Implementation Method 1
the sensor strand and the core strand bind complementarily to form a sensor duplex
Implementation Method 2
the guide strand and the core strand bind complementarily to form a RNAi duplex
Implementation Method 3
programmable, conditionally activated small interfering RNA (Cond-siRNA) construct
Data Source
AI summary
Disclosed herein are programmable, conditionally activated small interfering RNA constructs (Cond-siRNAs) and methods of making and using the same as therapeutic agents. The Cond-siRNA comprises a sensor strand, a core strand, and a guide strand, which crossover to form a sensor duplex and a RNAi duplex attached to each other to form a single structure. Upon binding an input strand to the sensor strand, the Cond-siRNA is activated and releases RNAi targeting a desired gene.