Prime Editing CRB1 Gene Correction Retinal Dystrophy
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Solution Overview
Problem
Current treatments lack effective methods for addressing mutations in the Crumbs homologue-1 (CRB1) gene, which cause progressive retinal dystrophies such as Leber congenital amaurosis 8 and retinitis pigmentosa, leading to severe visual impairment and blindness, with unclear pathogenesis and no available treatments.
Innovation Solution
The development of systems and methods utilizing prime editing technology, involving a Cas protein, reverse transcriptase, and specific RNA polynucleotides to modify the CRB1 gene by introducing base substitutions, deletions, or additions, targeting CRB1 mutations in cells, including retinal cells, to correct or insert mutations, thereby treating or preventing associated diseases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If prime editing technology is used to correct CRB1 mutations, then editing efficiency is improved, but delivery specificity to retinal cells becomes more difficult to achieve
Solution Approach 1:
The prime editing system is divided into separate functional components: a delivery vehicle (such as AAV vector) that can be optimized for retinal cell tropism, and editing components (Cas protein, pegRNA) that can be independently designed for specific CRB1 mutation targeting. This segmentation allows optimization of delivery specificity without compromising editing efficiency.
Solution Approach 2:
The patent applies local quality by designing delivery vehicles with tissue-specific tropism for retinal cells and using mutation-specific pegRNAs that target particular CRB1 mutation sites. The editing components are localized to the retinal genome through combination of retinal-tropic delivery and sequence-specific guide RNAs, achieving both efficient delivery and precise editing at the desired location.
2Reliability
If CRB1 mutations are corrected in retinal cells, then vision function is improved, but off-target effects and safety issues worsen
Solution Approach 1:
The patent replaces traditional CRISPR-Cas9 double-strand break mechanisms with prime editing, which uses a template-directed repair mechanism that does not require DNA double-strand breaks. This substitution reduces off-target effects and genomic instability while maintaining the ability to correct CRB1 mutations and restore vision function.
Solution Approach 2:
The patent uses an intermediary mechanism (prime editing with template-directed repair) between the delivery vehicle and the target mutation. The pegRNA serves as an intermediary that guides the Cas protein to the target site and provides the template for precise editing, mediating the correction process to minimize off-target effects while achieving reliable vision function restoration.
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
Achieves high editing efficiencies in correcting CRB1 patient mutations, demonstrating potential for treating or preventing retinal dystrophies by modifying the CRB1 gene, offering a promising therapeutic approach for severe visual impairments.
Implementation Method 1
The development of systems and methods utilizing prime editing technology, involving a Cas protein, reverse transcriptase, and specific RNA polynucleotides to modify the CRB1 gene by introducing base substitutions, deletions, or additions
Implementation Method 2
one or more RNA polynucleotides comprising a spacer sequence and an extension sequence comprising a primer binding sequence (PBS) and a reverse transcriptase template (RTT) sequence
Data Source
AI summary
The present disclosure provides systems, methods, and compositions for modifying the crumbs homologue-1 gene. Particularly the present disclosure provides systems, methods, and compositions for prime editing insertion or correction of mutations in the crumbs homologue-1 gene.