HTT Exon 1 sgRNA Design for Efficient Huntington's Gene Knockout
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Solution Overview
Problem
Current CRISPR/Cas-based HTT knockout strategies for Huntington's disease suffer from low gene editing efficiency and limited improvement in disease deficits, with short time-scale treatment effects.
Innovation Solution
Development of an sgRNA with specific nucleotide sequences (SEQ ID NO: 1 or SEQ ID NO: 2) for the CRISPR/Cas system, delivered via AAV9 vector to the striatum and cortical regions of the brain, achieving high efficiency in HTT gene knockout and significant improvement in disease phenotypes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR/Cas-based HTT knockout strategies are used, then HTT gene expression can be suppressed, but gene editing efficiency is low and treatment effects are limited
Solution Approach 1:
The patent optimizes the sgRNA nucleotide sequence parameters (specific sequences shown in SEQ ID NO: 1 or SEQ ID NO: 2) to enhance binding affinity and specificity to the HTT gene, thereby improving gene editing efficiency and overcoming the low efficiency limitation of conventional CRISPR/Cas strategies
Solution Approach 2:
The patent uses AAV9 vector as an intermediary delivery system to transport the optimized sgRNA to the striatum and cortical regions of the brain, enabling efficient and targeted delivery that overcomes the limitation of direct injection methods and achieves sustained treatment effects
2Reliability
If CRISPR/Cas system is delivered to brain regions, then HTT protein expression can be reduced, but delivery complexity increases
Solution Approach 1:
The patent employs AAV9 viral vector as an intermediary carrier that naturally tropes to brain tissue, particularly striatum and cortex regions, simplifying the delivery process compared to direct injection methods while achieving reliable HTT protein expression reduction through systemic or localized administration
Solution Approach 2:
The AAV9 vector system utilizes its inherent biological properties (tissue tropism, transduction efficiency) to autonomously navigate to target brain regions and deliver the sgRNA, reducing the need for complex external delivery infrastructure and procedures
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 sgRNA achieves approximately 90% reduction in HTT protein expression, improving HD-related phenotypic defects and demonstrating long-term treatment effects.
Implementation Method 1
CRISPR-Cas9 enables the editing of targeted genes using Cas9 nucleases with guide RNA. When both Cas9 protein and single-stranded guide RNA (sgRNA) are expressed in cells, sgRNA binds to specific sequences on the genome, recruiting Cas9 protein and generating DNA double-strand breaks
Implementation Method 2
delivered via AAV9 vector to the striatum and cortical regions of the brain
Data Source
AI summary
The present disclosure relates to an sgRNA and its application in the preparation of a product for the treatment of Huntington's disease. The present disclosure was designed and screened to obtain an sgRNA targeting exon 1 of the human HTT gene as shown in SEQ ID NO: 1 or SEQ ID NO: 2. The CRISPR/Cas9 system mediated HTT gene knockout strategy based on this sgRNA and its high homologue sgRNA can efficiently knock out the human Huntingtin gene and achieve gene therapy for Huntington's disease.