Split Intein Vector System for DMD Genomic Correction
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Solution Overview
Problem
Current therapies for Duchenne muscular dystrophy caused by frameshift mutations, such as exon deletions, offer only temporary and limited efficacy, and there is a need for a more efficient and permanent genomic correction method.
Innovation Solution
A vector system comprising two vectors, each encoding a fragment of an endonuclease fused to a split intein, is used to excise specific exons, restoring the reading frame of the dystrophin gene by associating into a functional intein and forming a functional endonuclease, specifically targeting and removing the mutated sequence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If antisense oligonucleotide (AON)-mediated exon skipping is used to reframe DMD transcripts, then the reading frame can be restored, but the efficacy is only temporary and limited
Solution Approach 1:
The patent replaces the mechanical/AON-based exon skipping approach with an endonuclease-based genomic editing system. Instead of using oligonucleotides to induce exon skipping, the invention uses endonucleases to precisely excise the mutated exon from the genome, creating a permanent correction that is replicated in all subsequent cell divisions.
Solution Approach 2:
The endonuclease system performs preliminary genomic correction by excising the mutated exon before protein synthesis occurs. This preliminary genetic modification ensures that all subsequent transcription and translation events produce correctly framed dystrophin, providing long-lasting efficacy rather than temporary suppression.
2Reliability
If endonuclease-based gene editing is used to correct genomic mutations, then permanent correction can be achieved, but the delivery system complexity increases
Solution Approach 1:
The patent divides the endonuclease system into multiple modular components delivered by separate vectors. Each vector carries specific elements (endonuclease gene, guide RNA, etc.), allowing independent optimization and controlled co-delivery. This segmentation reduces individual vector complexity while achieving the combined function of permanent genomic correction.
Solution Approach 2:
The patent uses guide RNA as an intermediary that directs the endonuclease to the specific mutated exon. This intermediary component simplifies the system by providing sequence-specific targeting without requiring complex protein-protein interactions or sophisticated delivery mechanisms, enabling precise genomic editing through a relatively simple RNA-mediated addressing system.
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 successfully restores dystrophin expression in muscles, including the heart, improves survival, and reduces arrhythmogenic vulnerability in DMD models, demonstrating a more efficient and permanent correction of the genetic mutation.
Implementation Method 1
the first fragment and the second fragment of the intein are capable of associating into a functional intein, wherein the functional intein is capable of ligating the first and the second fragment of the endonuclease to form a functional endonuclease
Implementation Method 2
the first gRNA binds to a region, which is located 5′ to a sequence of interest comprised in a nucleic acid sequence in the genome, preferably DNA, of a target cell, wherein the second gRNA binds to a region located 3′ to the sequence of interest
Implementation Method 3
the functional endonuclease is capable of excising the sequence of interest
Data Source
AI summary
The present invention relates a vector system and a vector system for use in a method of treating a disease, each comprising a first vector and a second vector. The present invention further relates to the first vector, the second vector and a combination of the first vector and the second vector. In addition, the present invention relates to a pharmaceutical composition comprising the vector system of the invention or the combination of the invention.