Base Editing Dystrophin Splice Sites for Exon Reframing
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Solution Overview
Problem
Current genome editing methods for Duchenne Muscular Dystrophy (DMD) rely on generating double-stranded breaks, leading to random insertions and deletions, which are inefficient and unpredictable, and do not effectively correct exon deletion mutations without introducing double cuts in the genome.
Innovation Solution
The use of adenine base editors (ABEs) and prime editors, which modify splice sites to restore the open reading frame of the dystrophin gene by inducing precise exon skipping or reframing without generating double-stranded breaks, utilizing a split-intein dual AAV system for in vivo delivery and CRISPR-Cas9 nickases to achieve targeted gene editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-Cas9 generates double-stranded breaks to introduce INDELs for reframing, then the ORF can be restored, but random insertions and deletions occur leading to unpredictable outcomes and off-target effects
Solution Approach 1:
The patent replaces the mechanical double-stranded break system with a chemical base substitution system. Adenine base editors convert specific adenine bases to guanine bases through deamination and repair mechanisms, eliminating the need for DSBs and the associated random INDELs and off-target effects while achieving precise reframing of the dystrophin gene ORF
Solution Approach 2:
The patent applies local quality by targeting specific adenine bases at precise locations within the dystrophin gene using guide RNAs. Each base editor is directed to a specific site to introduce a single nucleotide change that reframes the ORF, rather than creating random changes across the genome, thus achieving high precision with minimal off-target effects
2Productivity
If multiple sgRNAs are used to introduce double-cuts for exon removal, then larger INDELs can be achieved, but the complexity of the procedure increases and DSBs are still generated
Solution Approach 1:
The patent replaces the complex multi-sgRNA DSB system with a simpler base editing approach. Instead of coordinating multiple Cas9 proteins and guide RNAs to create DSBs, the system uses single or few base editors with corresponding guide RNAs to achieve precise nucleotide substitutions that refram the ORF, significantly reducing procedural complexity
Solution Approach 2:
The patent segments the editing function into modular base editor components that can be independently designed and deployed. Each base editor-gRNA complex acts as an independent unit targeting a specific site, allowing flexible combination of multiple editors if needed without the coordination complexity of multi-sgRNA DSB systems
3Reliability
If DSBs are generated through single-cut or double-cut strategies, then exon skipping can be achieved, but random INDELs are introduced reducing predictability of the outcome
Solution Approach 1:
The patent substitutes the unpredictable DSB repair mechanism with a controlled base substitution mechanism. Adenine base editors reliably convert adenine to guanine at specific sites through enzymatic deamination followed by cellular repair processes, ensuring predictable outcomes without the randomness inherent in NHEJ-mediated DSB repair
Solution Approach 2:
The patent changes the fundamental parameter of the editing mechanism from physical breakage (DSBs) to chemical modification (base substitution). This parameter change transforms the editing process from a stochastic DSB repair outcome to a deterministic base conversion outcome, significantly improving both predictability and precision
Data Source
AI summary
Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by the lack of dystrophin, which maintains muscle membrane integrity. Provided herein are methods of using adenine base editor (ABE) to modify splice sites of the dystrophin gene, causing skipping or refraining of common DMD exon deletion mutations, restoring dystrophin expression. Also provided herein are methods of using prime editing to reframe the dystrophin open reading frame and restore dystrophin expression.