Cas12f1 CEP290 Editing for AAV-Deliverable LCA10 Treatment
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Solution Overview
Problem
Current treatments for Leber congenital amaurosis type 10 (LCA10), an autosomal recessive disease caused by a biallelic loss-of-function mutation in the CEP290 gene, are limited due to the large size of the normal CEP290 gene exceeding packaging capacity of adeno-associated virus (AAV), and current treatments are not effective, and the CRISPR/Cas9 system faces challenges in gene editing efficiency and viral vector loading.
Innovation Solution
A CRISPR/Cas12f1 system is developed, comprising guide RNAs and a Cas12f1 protein, designed to target and modify the CEP290 gene, with enhanced double-stranded DNA cleavage activity, allowing for efficient removal or inversion of the 128 base pair cryptic exon, and is suitable for use in viral vectors like AAV.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If CRISPR/Cas9 system is used for gene editing, then gene editing capability is achieved, but viral vector loading is limited due to large protein size
Solution Approach 1:
The patent extracts the essential gene editing function from the bulky Cas9 protein and implements it through a miniaturized Cas12f1 protein that performs the same function with significantly reduced size, enabling efficient packaging into viral vectors
Solution Approach 2:
The patent changes the size parameter of the Cas protein from Cas9 (large) to Cas12f1 (miniaturized), transforming the physical characteristic to resolve the packaging limitation while maintaining editing functionality
2Ease of manufacture
If AAV packaging is used for gene replacement therapy, then therapeutic delivery is achieved, but packaging capacity is exceeded due to large CEP290 gene size
Solution Approach 1:
The patent extracts only the problematic region (cryptic exon) from the CEP290 gene for targeted removal, rather than delivering the entire large gene, thereby reducing the payload size below AAV packaging capacity limits
Solution Approach 2:
The patent segments the CEP290 gene into functional regions, identifying and isolating the cryptic exon (128 bp) as a separate target for removal, allowing selective editing without requiring delivery of the complete gene sequence
3Reliability
If CRISPR/Cas9 is used for LCA10 treatment, then treatment approach is established, but gene editing efficiency is insufficient
Solution Approach 1:
The patent changes the Cas protein parameter from Cas9 to miniaturized Cas12f1, which inherently provides enhanced double-stranded DNA cleavage activity, thereby improving gene editing efficiency and treatment reliability
Solution Approach 2:
The patent replaces the Cas9-based editing mechanism with a Cas12f1-based mechanism that utilizes multiple cleavage and repair processes, substituting the mechanical action to achieve superior editing outcomes
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 CRISPR/Cas12f1 system effectively addresses the limitations of CRISPR/Cas9 by enhancing gene editing efficiency through multiple cleavage and repair processes, enabling effective treatment of LCA10 by restoring normal CEP290 gene expression.
Implementation Method 1
the guide region has a guide sequence that forms a complementary bond with a target sequence present in the CEP290 gene
Implementation Method 2
enhancing double-stranded DNA cleavage activity, allowing for efficient removal or inversion of the 128 base pair cryptic exon
Data Source
AI summary
The present invention relates to LCA10 treatment using a CRISPR/Cas12f1(Cas14a1) system. In particular, the present invention relates to a composition comprising a CRISPR/Cas12f1(Cas14a1) system for LCA10 treatment, and to a treatment method using same. In addition, the present invention relates to a composition comprising a CRISPR/Cas12f1(Cas14a1) system for artificially manipulating the CEP290 gene, and to a method of editing the CEP290 gene.