Prime Editing Correcting PRPH2 Splice Site Mutations
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Solution Overview
Problem
Current treatments for PRPH2-mediated inherited retinal diseases are inadequate, as traditional base editing is inefficient for correcting c.828+3A>T and c.828+1G>T mutations, and existing methods face challenges with precision and off-target effects.
Innovation Solution
The use of prime editing systems involving Cas9 nickase, reverse transcriptase, and specific guide RNAs to correct c.828+3A>T, c.828+1G>A, and c.828+2T>C splice site mutations in the peripherin-2 gene, enabling precise nucleotide substitutions without prior knowledge of mutation presence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional base editing is used to correct PRPH2 mutations, then some base transitions can be achieved, but it cannot correct c.828+3A>T and c.828+1G>T mutations and has limited precision
Solution Approach 1:
The patent changes the editing mechanism from traditional base editing to prime editing, which uses a prime editor (Cas9 nickase fused to reverse transcriptase) and pegRNA to enable precise insertion, deletion, and substitution of nucleotides. This parameter change in the editing system allows correction of previously uncorrectable mutations like c.828+3A>T and c.828+1G>T while achieving higher precision through the primer-binding sequence mechanism that restricts editing to the intended target site.
2Productivity
If prime editing is used to correct multiple splice site mutations, then correction efficiency and precision are improved, but the system complexity increases
Solution Approach 1:
The patent employs a universal prime editing system that can correct multiple different splice site mutations (c.828+3A>T, c.828+1G>A, c.828+2T>C) using the same core components: Cas9 nickase, reverse transcriptase, and pegRNA. The pegRNA can be customized with different spacer sequences and reverse transcription templates to target various mutations, making the system multi-functional and highly efficient without requiring separate editing systems for each mutation type.
3Reliability
If existing editing methods are used, then some mutations can be addressed, but off-target effects and lack of precision remain problematic
Solution Approach 1:
The patent introduces a primer-binding sequence (PBS) as an intermediary element in the pegRNA that mediates precise binding to the target DNA site. The PBS anneals to the complementary sequence adjacent to the mutation site, positioning the reverse transcription template exactly where needed. This intermediary mechanism ensures that editing occurs only at the intended target site, eliminating off-target effects and greatly improving treatment reliability.
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 allows for efficient and precise correction of PRPH2 mutations, potentially restoring canonical mRNA splicing and preventing aberrant protein production, offering a therapeutic option for inherited retinal diseases.
Implementation Method 1
a reverse transcriptase; 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
Implementation Method 2
Cas protein is Cas9 or a variant or fragment thereof. In some embodiments, the Cas protein is a Cas9 nickase
Data Source
AI summary
The present disclosure provides systems, methods, and compositions for prime-editing modification of c.828 splice site mutations in the peripherin-2 gene. Particularly the present disclosure provides systems, methods, and compositions for correcting one or more disease-causing splice site mutations selected from: c.828+3A>T, c.828+1G>A, c.828+2T>C, c.828+1G>T, and combinations thereof.