Protease-Activated siRNA for Mutation-Resistant Gene Silencing
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Solution Overview
Problem
Current biologically active molecules, such as siRNA and PNA, face challenges in selectively targeting virus-, bacteria-, parasite-infected cells, and tumor cells due to instability and rapid mutation of target proteases, leading to ineffective inhibition of viral, bacterial, or tumor growth.
Innovation Solution
Biologically active molecules comprising a protease inhibitor and peptide-inhibited siRNA, PNA, or RNA that can be activated by the target protease, even in the presence of mutations, to suppress gene expression and inhibit cellular activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If protease inhibitors are used to target virus-, bacteria-, parasite-infected cells and tumor cells, then the biological activity of target proteases can be inhibited, but mutations in the target proteases can reduce binding affinity and render the inhibitors ineffective
Solution Approach 1:
The invention divides the therapeutic approach into two independent components: a protease inhibitor and a peptide-inhibited siRNA/PNA/RNA molecule. Each component targets the same protease but through different mechanisms. The protease inhibitor binds to the active site, while the peptide-inhibited nucleic acid molecule contains a peptide sequence that mimics the protease's natural substrate. When the protease mutates and loses affinity for the inhibitor, it can still recognize and cleave the peptide sequence in the siRNA/PNA/RNA, activating the gene silencing mechanism. This segmentation allows the system to maintain effectiveness even when one component becomes less effective due to mutation.
Solution Approach 2:
The invention changes the parameter of molecular stability by protecting the siRNA/PNA/RNA molecules through peptide inhibition. The peptide sequence acts as a protective mask that prevents premature activation of the nucleic acid molecules. Only when the target protease cleaves the peptide sequence does the siRNA/PNA/RNA become active. This parameter change from stable-but-inactive to activated-at-target ensures that the molecules remain stable during delivery but become effective precisely when and where needed, overcoming the limitation of protease mutation.
2Reliability
If siRNA and PNA molecules are introduced directly into cells via transfection reagents and electroporation, then gene expression can be suppressed, but the molecules are relatively unstable and degradation occurs
Solution Approach 1:
The invention applies preliminary protection to the siRNA/PNA/RNA molecules by conjugating them with a peptide sequence that is recognized by the target protease. This peptide acts as a protective cloak during delivery, preventing premature degradation by cellular nucleases. The protection is built into the molecule structure before introduction into the cell. Only after the protease cleaves the peptide does the nucleic acid molecule become active. This preliminary protective action ensures the molecules survive the delivery process and reach their target intact.
3Reliability
If biologically active substances are administered in vivo, then systemic effects occur, but selective introduction into target cells is not sufficiently specific
Solution Approach 1:
The invention uses the target protease itself as an intermediary to deliver the therapeutic effect selectively. The protease inhibitor and peptide-inhibited siRNA/PNA/RNA molecules are designed to interact specifically with the target protease that is overexpressed or mutated in virus-, bacteria-, parasite-infected cells and tumor cells. When administered in vivo, only cells expressing the target protease will activate the peptide-inhibited molecules through cleavage. This intermediary mechanism ensures that even though the substances are distributed systemically, only the intended target cells are affected, minimizing off-target effects.
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 solution ensures effective inhibition of viral, bacterial, or tumor cell replication and growth by activating the siRNA or PNA molecules, even if protease inhibitors lose binding affinity due to mutations, thereby reducing gene expression and inducing apoptosis or necrosis.
Implementation Method 1
peptide-inhibited siRNA, PNA or RNA, the peptide bond of which is broken by the at least one specific target protease for the purpose of activating the peptide-inhibited siRNA, PNA or RNA
Implementation Method 2
the reading of a gene and the production of an mRNA are not prevented, but rather, in the case of siRNA, a cell's own mechanism is initiated that breaks down the target mRNA
Implementation Method 3
Finally, as described above, the formation of a specific protein is suppressed without affecting the expression of other genes (post-transcriptional gene silencing)
Data Source
Figure 1a~1c
Figure 2~3
AI summary
2.1 The aim of the invention is to effectively inhibit virus-, bacteria-, or parasite-infected cells and tumor cells in a targeted manner, even in the case of mutations. 2.2 According to the invention, biologically active molecules are administered, said biologically active molecules comprising at least one protease inhibitor (3) for at least one specific target protease (2) of the virus-, bacteria-, or parasite-infected cells and/or tumor cells (1) and at least one peptide-inhibited siRNA, PNA, or RNA (5), the peptide bond (4) of which is broken up by the at least one target protease (2) for the purpose of activating the peptide-inhibited siRNA, PNA, or RNA (5). 2.3. Said molecules are used, for example, to influence the gene expression of diseased and infected organs or cells.