Non-Coding RNA Specificity Modification via DNA Editing
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Solution Overview
Problem
Current gene therapy and RNA interference technologies face limitations in efficiently delivering RNA into cells and achieving complete repression of gene function, particularly for diseases requiring precise regulation, due to issues like mutagenesis, transient nature of siRNA, and limited silencing amplification mechanisms.
Innovation Solution
A method involving DNA or RNA editing agents that redirect the silencing specificity of non-coding RNA molecules to target RNAs of interest, using CRISPR/Cas systems and other editing tools to introduce base editing and confer silencing activity, allowing for precise modification of gene expression in eukaryotic cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If RNA interference (RNAi) is used to repress gene function, then gene expression can be reduced, but complete repression is not achieved due to transient nature of siRNA and lack of silencing amplification mechanisms
Solution Approach 1:
The patent applies preliminary action by integrating the RNAi machinery components (Pol III recognition sequences, terminator sequences) directly into the genomic DNA before the need for gene repression. This creates a self-sustaining system where the cell continuously produces siRNA from the integrated construct, eliminating the need for repeated transient transfections and achieving long-term, complete gene silencing.
Solution Approach 2:
The invention implements self-service by designing an autonomous RNAi system where the integrated Pol III transcript includes all necessary elements (promoter, coding sequence, terminator) to generate functional siRNA continuously. The system serves itself by utilizing the cell's existing Pol III transcription machinery and RNA processing pathways,无需 external intervention for sustained gene repression.
2Reliability
If viral gene therapy is used to restore gene function, then missing gene function can be restored, but mutagenesis at integration site and dysregulated transgene expression may occur
Solution Approach 1:
The patent uses an intermediary approach by employing Pol III-driven RNA transcription as a mediator between the integrated DNA and the desired gene silencing effect. Instead of directly integrating functional genes (viral gene therapy), the system integrates a DNA construct that produces RNA molecules which then mediate the silencing of target genes through the RNAi pathway, avoiding direct protein expression and its associated risks.
Solution Approach 2:
The invention replaces the mechanical system of viral integration and protein expression with an RNA-based mechanism. By substituting direct gene restoration (mechanical DNA integration) with RNA-mediated silencing, the system achieves gene function modulation without the harmful effects of viral mutagenesis and dysregulated transgene expression.
3Productivity
If delivery agents are used to enhance RNA endocytosis, then RNA delivery into cells is improved, but less than 0.01% escapes from endosomes and is biologically active
Solution Approach 1:
The patent replaces the mechanical delivery system (exogenous RNA transfection requiring endocytosis and endosomal escape) with a biological synthesis system. By integrating the RNAi machinery into the genome and using Pol III to transcribe siRNA directly in the nucleus, the system bypasses the need for endocytosis and endosomal escape entirely, achieving near 100% delivery efficiency and biological activity.
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
This approach enables efficient and precise modification of gene expression, effectively silencing target genes associated with diseases, including those with high copy numbers or involved in apoptosis, thereby providing a broader therapeutic scope for genetic disorders.
Implementation Method 1
allowing the cell's endogenous DSB repair machinery to fix the break (such as by non-homologous end-joining (NHEJ) or homologous recombination (HR)) in which the latter can allow precise nucleotide changes to be made to the DNA sequence
Implementation Method 2
RNA interference (RNAi), which mediates repression of defective genes by knockdown of the target mRNA
Data Source
AI summary
A method of modifying a gene encoding or processed into a non-coding RNA molecule having no RNA silencing activity in a eukaryotic cell, wherein the gene encoding or processed into the non-coding RNA molecule is positioned in a coding gene, is disclosed. The method comprising introducing into the eukaryotic cell a DNA editing agent conferring a silencing specificity of the non-coding RNA molecule towards a target RNA of interest. Target RNA of interest include, for example, a transcript of a gene selected from the group consisting of a housekeeping gene, a dominant gene, a gene comprising a high copy number, and a gene associated with cell apoptosis. Methods comprising DNA or RNA editing agents which elicit base editing are also disclosed.