Trans-splicing Ribozyme for Rhodopsin Transcript Replacement
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Solution Overview
Problem
Current treatments for autosomal dominant retinitis pigmentosa (ADRP) caused by rhodopsin gene mutations, such as the P23H mutation, are limited, as existing methods like CRISPR/Cas9 and siRNA/antisense RNA can only target single gene mutations and introduce foreign proteins, leading to immune responses, and do not restore the wild-type RHO gene expression effectively.
Innovation Solution
Development of a trans-splicing ribozyme system that targets rhodopsin transcripts, capable of deleting mutant transcripts and replacing them with wild-type RHO transcripts, using a non-viral gene delivery system to induce wild-type RHO gene expression, thereby addressing multiple mutations and minimizing immune response risks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional RNAi approaches are used to target retinal diseases, then gene expression can be suppressed, but the approaches lack specificity for rhodopsin transcripts and cannot distinguish between different transcript variants
Solution Approach 1:
The guide RNA is divided into two separate modules: a CRISPR RNA (crRNA) component that provides transcript-specific targeting through its spacer sequence, and a trans-activating crRNA (tracrRNA) component that provides structural functionality. This segmentation allows independent optimization of specificity (crRNA) and structural requirements (tracrRNA), resolving the contradiction between achieving high transcript specificity and managing design complexity.
Solution Approach 2:
A trans-splicing ribozyme acts as an intermediary enzyme that catalyzes the joining of the crRNA and tracrRNA modules. This ribozyme-mediated trans-splicing process enables the formation of a functional guide RNA complex with high specificity for rhodopsin transcripts while using standardized, well-characterized molecular components, thus reducing overall system complexity.
2Reliability
If RNA interference is applied to treat retinal diseases, then therapeutic effect can be achieved, but off-target effects and lack of selectivity for specific transcript variants occur
Solution Approach 1:
The crRNA component is designed with a spacer sequence that is locally optimized to match specific rhodopsin transcript variants (e.g., differentiating between wild-type and mutant transcripts). This local sequence customization provides high selectivity for the target transcript while maintaining overall system reliability and minimizing off-target effects on non-target genes.
Solution Approach 2:
Instead of using traditional siRNA that relies on imperfect mismatch discrimination, this invention uses a CRISPR-based system where the spacer sequence is designed to be perfectly complementary to the target transcript. The inversion approach is to use a different molecular mechanism (CRISPR-Cas9 guided cleavage) rather than relying on RNAi's natural mismatch tolerance, thereby achieving higher selectivity and reduced off-target effects.
3Adaptability or versatility
If standard RNAi mechanisms are used, then gene silencing can be achieved, but the system cannot be easily adapted to target specific transcript variants of rhodopsin
Solution Approach 1:
The tracrRNA component serves multiple functions: it provides structural scaffolding for the guide RNA complex, recruits the Cas9 enzyme, and participates in trans-splicing with the crRNA. This universal, multi-functional design allows the same tracrRNA sequence to be used with different crRNA variants targeting different rhodopsin transcripts, thereby achieving high adaptability while maintaining ease of manufacture through standardized components.
Solution Approach 2:
The crRNA and tracrRNA are designed as pre-formed, standardized modules that can be independently synthesized and stored. The trans-splicing ribozyme is also a defined molecular entity. This preliminary preparation of functional modules allows rapid adaptation to different transcript variants by simply changing the crRNA spacer sequence, without requiring de novo design of the entire guide RNA, thus maintaining ease of manufacture while achieving high versatility.
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
The trans-splicing ribozyme system effectively replaces mutant rhodopsin transcripts with wild-type transcripts, providing a curative treatment for retinitis pigmentosa by restoring normal rhodopsin function and minimizing toxicity and immune response concerns.
Implementation Method 1
a trans-splicing ribozyme, and a capped connection RNA (cConnection RNA). The trans-splicing ribozyme may be derived from the hepatitis delta virus (HDV) ribozyme
Implementation Method 2
a Cas9 endonuclease, and a single-stranded DNA (ssDNA) repair template
Data Source
Figure 1~2
Figure 3a
Figure 3b
AI summary
The present invention relates to a trans-splicing ribozyme targeting a rhodopsin transcript and its use for the treatment and/or prophylaxis of retinitis pigmentosa.