Allele-Specific RNA Reduction via CRISPR Poison Exon Editing
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Solution Overview
Problem
Human diseases with dominant negative inheritance patterns pose challenges for treatment using gene therapy methods, particularly when a wild-type allele is crucial for normal function, necessitating allele-specific gene editing.
Innovation Solution
Utilizing CRISPR-Cas systems with guide nucleic acids to introduce a poison exon or mutation in a target nucleic acid, specifically at single-nucleotide polymorphisms, to create a premature stop codon in the pathogenic allele, triggering nonsense-mediated mRNA decay and reducing the level of toxic gain-of-function proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If gene therapy methods are used to treat dominant negative inheritance diseases, then the pathogenic allele can be targeted, but the wild-type allele may be inadvertently affected, compromising normal function
Solution Approach 1:
The patent applies local quality by designing guide RNAs with sequences that are specific to the pathogenic allele's unique genetic variations (such as CAG repeats or SNPs). This allows the CRISPR-Cas system to distinguish and target only the affected allele while leaving the wild-type allele untouched, thereby achieving allele-specific therapy without compromising normal gene function
Solution Approach 2:
The patent segments the therapeutic approach by using multiple guide RNAs with different target sequences within the pathogenic allele. This segmentation allows for precise targeting of specific regions (such as the expanded CAG repeat region in Huntington's disease) while maintaining specificity and avoiding off-target effects on the wild-type allele
2Reliability
If CRISPR-Cas systems are used to introduce poison exons or stop codons, then toxic gain-of-function proteins can be reduced, but the complexity of the gene editing process increases
Solution Approach 1:
The patent applies preliminary action by designing and introducing poison exons or stop codons at specific locations within the pathogenic allele before they can produce toxic proteins. The guide RNAs are designed to target sequences upstream or within the pathogenic region, allowing the CRISPR-Cas system to introduce disabling mutations that prevent toxic protein synthesis in advance
Solution Approach 2:
The patent converts the harmful pathogenic sequences (such as expanded CAG repeats or mutant SNPs) into beneficial targets by designing guide RNAs that specifically recognize these abnormal sequences. The very genetic features that cause the disease (repeats, mutations) become the precise targets for therapeutic intervention, allowing the system to exploit the pathogenic allele's uniqueness for selective targeting
3Measurement precision
If allele-specific gene editing is performed at single-nucleotide polymorphisms, then specificity is improved, but the difficulty of detecting and measuring the target increases
Solution Approach 1:
The patent uses guide RNAs as intermediaries that bridge the CRISPR-Cas system and the single-nucleotide polymorphism targets. The guide RNAs are designed with sequences that are complementary to the specific mutant nucleotide sequence, allowing the system to detect and target single-nucleotide differences with high precision. The guide RNA acts as a mediator that translates the subtle nucleotide difference into a recognizable target for the Cas effector
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
Effectively reduces the level of toxic proteins by at least 5-80% through allele-specific targeting, ensuring the wild-type allele remains functional.
Implementation Method 1
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems comprise a CRISPR-associated (Cas) effector polypeptide and a guide nucleic acid. Such CRISPR-Cas systems can bind to and modify a targeted nucleic acid.
Implementation Method 2
nonsense-mediated mRNA decay (NMD). An mRNA transcript with a premature stop codon (right) is flagged by the NMD surveillance pathway as erroneous and is tagged for RNA degradation.
Data Source
AI summary
The present disclosure provides methods for reducing the level of an RNA transcript from a target nucleic acid. The present disclosure provides methods of treating a disease that results from or is caused by a toxic gain-of-function protein. The present disclosure provides systems and compositions for carrying out a method of the present disclosure.