Mitochondrial Targeted RNA Expression System for Disease Therapy
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Solution Overview
Problem
Current therapies are inadequate for addressing diseases caused by mutations in mitochondrial genes, particularly those affecting the electron transport chain, as they fail to effectively modulate mitochondrial function or inhibit mutant protein expression.
Innovation Solution
A mitochondrial-targeted RNA expression system (mtTRES) is developed, comprising specific sequences for targeting RNA molecules to mitochondria, including an RNA polymerase III promoter, non-coding mitochondrial leader sequences, and open reading frames or sequences that inhibit translation, allowing for the delivery of therapeutic RNAs to modulate mitochondrial function or repress mutant gene expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional gene therapy approaches are used to treat mitochondrial diseases, then therapeutic potential is limited, but the complexity of delivering functional RNAs to mitochondria remains high
Solution Approach 1:
The delivery system is segmented into distinct functional components: a nuclear export signal sequence for nuclear egress, a mitochondrial targeting sequence for mitochondrial import, and a functional RNA payload. This segmentation allows each component to be optimized independently for its specific function while working together as an integrated delivery system.
Solution Approach 2:
The patent employs intermediary sequences as molecular mediators between the cytosol and mitochondria. Specifically, the mitochondrial targeting sequence acts as an intermediary that recognizes mitochondrial import receptors and facilitates the transport of the functional RNA from the cytosolic compartment into the mitochondrial matrix, overcoming the complexity of direct delivery.
2Reliability
If RNA molecules are delivered to mitochondria for therapeutic effect, then mutant protein expression can be inhibited, but ensuring specific targeting to mitochondria without cytosolic expression is difficult
Solution Approach 1:
The RNA molecule is designed with local quality differentiation through compartment-specific sequences. The 5' end contains a nuclear export signal for selective nuclear egress, while the 3' end contains a mitochondrial targeting sequence for selective mitochondrial import. This local differentiation ensures the RNA is functional only in mitochondria, preventing cytosolic expression of mutant proteins.
Solution Approach 2:
Instead of attempting to prevent cytosolic expression through negative control mechanisms, the patent inverts the approach by designing positive control sequences that actively direct the RNA to mitochondria. The mitochondrial targeting sequence serves as a positive instruction set that overrides any potential cytosolic translation, ensuring exclusive mitochondrial localization.
3Productivity
If functional RNAs are expressed in mitochondria, then wild-type protein can be provided, but preventing translation in the cytosol is challenging
Solution Approach 1:
The RNA sequence is designed with local quality differences that create compartment-specific translation. The mitochondrial targeting sequence at the 3' end, combined with mitochondrial-specific translation initiation sequences, creates a local quality signature that is recognized only by mitochondrial ribosomes, preventing cytosolic translation while maintaining efficient mitochondrial protein synthesis.
Solution Approach 2:
The mitochondrial targeting sequence acts as an intermediary that mediates between the RNA payload and the mitochondrial translation machinery. It recognizes mitochondrial import receptors and facilitates interaction with mitochondrial ribosomes, while preventing recognition by cytosolic translation initiation factors, thus blocking harmful cytosolic translation.
Data Source
AI summary
Described herein is a mitochondrial-targeted RNA expression system (mtTRES) for delivery of RNA molecules to mitochondria. mtTRES vectors generate RNAs in vivo that are un-capped, non-polyadenylated, and actively directed to mitochondria. The disclosed vectors are capable of delivering either non-coding RNA molecules or RNA molecules encoding a protein of interest to the mitochondria. In particular, the disclosed vectors include (1) an RNAPIII initiation (promoter) sequence, (2) a non-coding leader sequence (NCL), (3) a mitochondrial translation initiation sequence and an ORF encoding a protein of interest, or a sequence encoding a non-coding RNA, and (4) an RNAPIII termination sequence.