Modified dsRNA Constructs Resolving Nuclease Vulnerability
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Classic siRNAs face limitations such as instability due to nuclease vulnerability, off-target effects, and reduced specificity in gene silencing, primarily due to their double-stranded nature and susceptibility to Dicer cleavage, which affects their therapeutic efficacy.
Innovation Solution
Development of blunt-ended double-stranded RNA constructs with 2'-modified ribose sugars at specific ends, resistant to Dicer cleavage, allowing for sequence-dependent target gene inhibition and improved stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If classic siRNA is used with double-stranded structure and 19-23 nucleotides length, then RNA interference activity is achieved, but nuclease vulnerability and spontaneous hydrolysis occur leading to instability
Solution Approach 1:
The patent applies parameter changes by modifying the chemical structure of ribonucleotides through 2'-O-methylation and replacing phosphodiester linkages with phosphorothioate linkages. These chemical parameter changes confer nuclease resistance while preserving RNA interference activity, directly resolving the contradiction between reliability and stability.
Solution Approach 2:
The patent creates composite oligonucleotide structures by combining modified nucleotides (2'-O-methyl ribonucleotides) with unmodified nucleotides, and combining different linkage types (phosphorothioate and phosphodiester). This composite approach provides both nuclease resistance and functional activity, resolving the stability-reliability contradiction.
2Reliability
If Dicer cleavage is utilized to generate 21-mer siRNA products, then RNAi activity is achieved, but heterogeneous mixture results with different biological activities and off-target effects
Solution Approach 1:
The patent applies preliminary anti-action by designing oligonucleotides with 2'-O-methyl modifications at positions that prevent Dicer recognition and cleavage. This preemptive modification blocks the formation of heterogeneous Dicer products, ensuring a single specific active species with defined target specificity and eliminating off-target effects.
Solution Approach 2:
The patent extracts the problematic Dicer cleavage step from the RNAi pathway by designing oligonucleotides that are resistant to Dicer processing. This removes the source of heterogeneity and off-target effects, allowing direct use of the designed sequence without Dicer-mediated fragmentation.
3Reliability
If longer oligonucleotides are used to increase binding activity to target RNA, then higher gene silencing activity is achieved, but overhangs cause instability and degradation
Solution Approach 1:
The patent applies parameter changes by extending the oligonucleotide length beyond the classic 19-23 nucleotides while incorporating 2'-O-methyl modifications and phosphorothioate linkages. These chemical parameter changes protect the extended regions from nuclease degradation, allowing longer sequences that provide enhanced binding activity without sacrificing stability.
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 constructs demonstrate enhanced target specificity, reduced off-target silencing, and increased stability, leading to more effective and predictable gene silencing with improved pharmacological properties.
Implementation Method 1
wherein the sense strand is 25, 26, 27, 28, 29 or 30 nucleotides in length, and an antisense strand having a 5'-end and a 3'-end, wherein the antisense strand is 25, 26, 27, 28, 29 or 30 nucleotides in length, for inhibiting expression of a SOD1 gene, wherein the 5'-end 12 nucleotides and the 3'-end 10 nucleotides of the sense strand are 2'-modified ribose sugars
Implementation Method 2
wherein the antisense strand hybridizes to the sense strand and to mRNA of the SOD1 gene
Implementation Method 3
said dsRNA is resistant to cleavage by Dicer
Implementation Method 4
the antisense strand associates with RISC, and (c) the dsRNA inhibits expression of the target gene in a sequence-dependent manner
Data Source
Figure 1
Figure 2
Figure 3
AI summary
The invention relates to improved RNAi constructs and uses thereof. The construct has a double stranded region of 19-49 nucleotides, preferably 25, 26, or 27 nucleotides, and preferably blunt-ended. The construct has selective minimal modifications to confer an optimal balance of biological activity, toxicity, stability, and target gene specificity. For example, the sense strand may be modified (e.g., one or both ends of the sense strand is/are modified by four 2'-O-methyl groups), such that the construct is not cleaved by Dicer or other RNAse III, and the entire length of the antisense strand is loaded into RISC. In addition, the antisense strand may also be modified by 2'-O-methyl group at the 2nd 5'-end nucleotide to greatly reduce off-target silencing. The constructs of the invention largely avoids the interferon response and sequence- independent apoptosis in mammalian cells, exhibits better serum stability, and enhanced target specificity.