Base Editing USH2A Gene for AAV Packaging and Ectopic Expression
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Solution Overview
Problem
Current gene therapies for USH2A-associated retinitis pigmentosa are challenging due to the large size of the USH2A coding sequence exceeding the packaging capacity of commonly used AAV viral delivery vectors, and existing treatments are not curative, requiring repeat dosing and having limited efficacy in reversing visual and hearing loss in Usher syndrome.
Innovation Solution
The use of CRISPR/Cas base editing approaches with fusion proteins containing deaminase and nucleic acid programmable DNA binding proteins, along with guide RNAs, to directly edit the USH2A gene locus, enabling targeted edits and increased stability and activity of the Usherin protein, thereby addressing the limitations of conventional gene therapies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If AAV viral delivery vectors are used to deliver USH2A gene therapy, then gene delivery can be achieved, but the large size of USH2A coding sequence exceeds the packaging capacity of AAV vectors
Solution Approach 1:
The patent extracts only the essential functional elements needed for gene therapy (promoter, partial coding sequence, polyA signal) and removes non-essential portions of the USH2A gene. This creates a minimized gene construct that fits within AAV packaging capacity while retaining therapeutic functionality.
Solution Approach 2:
The USH2A gene therapy construct is segmented into essential functional components (promoter region, coding sequence, polyA signal) that can be independently optimized and packaged. This segmentation allows the therapeutic gene to be delivered within AAV vector constraints.
2Reliability
If conventional gene therapies are used to treat USH2A mutations, then some efficacy can be achieved, but the treatments are not curative and require repeat dosing
Solution Approach 1:
The patent uses base editing to preemptively correct the pathogenic mutation in the USH2A gene before it can cause progressive damage. By establishing the correct gene sequence early, the treatment aims to provide lasting correction rather than temporary symptom management, eliminating the need for repeat dosing.
Solution Approach 2:
The patent replaces conventional gene addition therapies with base editing technology, substituting a mechanical correction approach that directly modifies the existing gene sequence. This substitution enables curative potential by correcting the root cause rather than adding compensatory genes.
3Ease of manufacture
If conventional viral-mediated therapies are used, then gene delivery can be achieved, but ectopic expression issues arise
Solution Approach 1:
The patent utilizes the cell's own endogenous USH2A gene regulatory elements (promoter, polyA signal) to control expression of the corrected gene sequence. This self-service approach ensures proper spatial and temporal expression patterns without requiring external viral regulatory elements that cause ectopic expression.
Solution Approach 2:
The endogenous USH2A gene regulatory regions serve multiple functions: they drive appropriate expression of the corrected gene, ensure proper tissue-specific expression patterns, and eliminate the need for separate viral regulatory elements. This multi-functionality resolves the ectopic expression problem while maintaining ease of delivery.
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 effectively reverses visual and hearing loss by restoring functional Usherin protein, offering a potentially curative treatment with reduced off-target effects and avoiding ectopic expression issues associated with conventional viral-mediated therapies.
Implementation Method 1
deaminating a cytosine nucleobase in the USH2A gene... deaminating an adenosine nucleobase in the USH2A gene
Data Source
AI summary
The disclosure provides methods of deaminating adenosine and cytosine bases in a target nucleic acid sequence in an USH2A gene comprising contacting the USH2A gene with a base editor in association with a guide RNA (gRNA). In some aspects, base editing is used to restore US2HA function by disrupting a splice site in the USH2A gene sequence to induce skipping of an exon containing a mutation, while in other embodiments, base editing is used to restore US2HA function by correcting a point mutation e.g., in an exon) so as to correct mutations. The disclosure also provides complexes of adenosine base editors and guide RNAs, and complexes of cytidine base editors and guide RNAs. The disclosure further provides pharmaceutical compositions and cells comprising these complexes. The disclosure also provides vectors encoding these complexes, base editors, and gRNAs. In some embodiments, the methods and compositions provided herein are used to treat Usher syndrome and autosomal recessive retinitis pigmentosa (arRP).