CRISPR CasX Gene Editing for Rhodopsin Mutation Correction
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Solution Overview
Problem
Current treatments for retinitis pigmentosa are inadequate, and there is a need for safe and permanent solutions to address the progressive neurodegenerative disorder caused by mutations in the rhodopsin gene, particularly the P23H mutation, which affects protein folding and function in photoreceptor cells.
Innovation Solution
The use of modified Class 2, Type V CRISPR proteins and guide nucleic acids to edit the RHO gene, allowing for passive entry into cells and targeting specific mutations, such as the P23H mutation, to correct or compensate for genetic defects, thereby promoting the expression of functional rhodopsin protein.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR/Cas systems are used to edit rhodopsin gene mutations, then genetic defects can be corrected to restore functional rhodopsin expression, but delivery into target photoreceptor cells remains challenging due to system size and complexity
Solution Approach 1:
The CRISPR system is divided into separate components: Class 2 Type V CRISPR protein (Cas12a/CasY) and guide RNA are delivered as distinct entities that assemble in the target cell. This segmentation allows each component to be optimized independently for delivery and function, reducing the complexity of delivering a single large complex system while maintaining gene editing effectiveness.
Solution Approach 2:
Adeno-associated virus (AAV) vectors serve as intermediaries to deliver the CRISPR components into photoreceptor cells. The AAV system acts as a mediator that overcomes the delivery barrier by utilizing viral mechanisms for efficient cellular entry and nuclear delivery, enabling the CRISPR system to reach its target without direct injection or complex delivery apparatus.
2Reliability
If conventional CRISPR systems are delivered into cells, then gene editing can be performed, but the large size of the system hinders efficient delivery and expression
Solution Approach 1:
The system uses Class 2 Type V CRISPR proteins (Cas12a, CasY) which are inherently smaller than conventional Cas9 proteins. The guide RNA is also optimized to be compact. This segmentation and size reduction of individual components enables efficient delivery via AAV vectors while maintaining complete gene editing functionality.
Solution Approach 2:
The patent utilizes Class 2 Type V CRISPR proteins which have different physical parameters (smaller size, different structural organization) compared to conventional Type II CRISPR-Cas9 systems. This parameter change in protein class and size allows the system to fit within delivery vector constraints while preserving cutting and editing capabilities.
3Reliability
If rhodopsin mutations are left uncorrected, then current treatments provide only symptomatic relief, but correcting the mutations requires sophisticated gene editing tools that are difficult to deliver to retinal cells
Solution Approach 1:
AAV vectors serve as the intermediary delivery vehicle that simplifies the administration process. By encapsulating the CRISPR components within AAV, the complex gene editing system can be delivered via established ocular injection routes (subretinal or intravitreal injection), making the treatment administrable by ophthalmologists without requiring complex surgical procedures or specialized equipment.
Solution Approach 2:
The CRISPR system is designed to be self-contained and self-activating within the target cell. Once delivered by AAV, the CRISPR protein and guide RNA automatically assemble and locate the rhodopsin gene mutation for correction without requiring additional external control or activation steps, simplifying the overall treatment protocol.
Data Source
AI summary
Provided herein are Class 2 Type V CRISPR:gNA systems comprising Class 2 Type V CRISPR polypeptides (e.g. CasX), guide nucleic acids (gNA), and optionally donor template nucleic acids useful in the modification of a RHO gene. The systems are also useful for introduction into cells, for example eukaryotic cells having mutations in the rhodopsin protein. Also provided are methods of using such systems to modify cells having such mutations and utility in methods of treatment of a subject with a RHO-related disease, such as retinitis pigmentosa.