APOA1 Allele-Specific CRISPR Knockout for Amyloidosis
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Solution Overview
Problem
Dominant genetic disorders caused by mutations in the APOA1 gene, such as amyloidosis, are challenging to treat due to the dominance of the mutant allele over the functional allele, leading to protein misfolding and tissue disruption.
Innovation Solution
Utilizing heterozygous single nucleotide polymorphisms (SNPs) to distinguish between mutant and functional alleles of the APOA1 gene, employing CRISPR nuclease and guide RNA molecules to selectively knock out the mutant allele and degrade the resulting mRNA, thereby allowing expression of the functional protein.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional treatment approaches are used for dominant genetic disorders, then the mutant allele continues to dominate and cause disease, but selective knockout of the mutant allele while preserving the functional allele is not achieved
Solution Approach 1:
The patent divides the targeting strategy into two distinct components: a CRISPR nuclease component and a guide RNA component. The guide RNA is further segmented to include both a target recognition sequence (20 nucleotides) and a polymorphism-specific sequence (8 nucleotides), allowing the system to separately address allele identification and cleavage functions, thereby achieving allele-specific knockout while managing system complexity
Solution Approach 2:
The patent introduces guide RNA as an intermediary molecule that mediates between the CRISPR nuclease and the target DNA. The guide RNA contains a polymorphism-specific sequence that recognizes the mutant allele's unique nucleotide sequence, acting as a selective intermediary that directs the nuclease only to the mutant allele while sparing the functional allele
2Manufacturing precision
If CRISPR nuclease and guide RNA are used to target the mutant allele, then selective knockout is achieved, but the complexity of the molecular system increases
Solution Approach 1:
The patent applies local quality by designing the guide RNA with a specific 8-nucleotide polymorphism-specific sequence that is localized to the target recognition region. This localized sequence feature provides high precision allele discrimination by matching only the mutant allele's unique polymorphism, while the rest of the CRISPR system remains relatively simple and well-established
Solution Approach 2:
The patent changes the nucleotide sequence parameter of the guide RNA to include the polymorphism-specific sequence that matches the mutant allele's unique genetic signature. This parameter change in the guide RNA sequence allows precise differentiation between mutant and functional alleles, achieving high manufacturing precision through sequence-specific targeting
3Object-affected harmful factors
If the mutant allele is knocked out, then protein misfolding is prevented, but the functional allele may be inadvertently affected
Solution Approach 1:
The patent incorporates feedback through the polymorphism-specific sequence in the guide RNA, which provides selective recognition based on the mutant allele's unique genetic signature. This feedback mechanism ensures that only cells carrying the specific mutant polymorphism are targeted, allowing prevention of protein misfolding while preserving cells with the functional allele through sequence-specific discrimination
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 effectively inactivates the mutant APOA1 allele, preventing protein misfolding and tissue disruption, offering a potential treatment for amyloidosis by selectively targeting and degrading the mutant allele while preserving the functional allele's expression.
Implementation Method 1
employing CRISPR nuclease and guide RNA molecules to selectively knock out the mutant allele
Implementation Method 2
an RNA molecule comprising a guide sequence targets only the mutant allele of the gene by targeting the nucleotide base present at a heterozygous SNP in the mutant allele of the gene
Data Source
AI summary
RNA molecules comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-1313 and compositions, methods, and uses thereof.