CRISPR-Mediated Repeat Expansion for Neurodegenerative Disease Models
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Solution Overview
Problem
There is a lack of effective animal models that accurately recapitulate repeat expansion diseases such as neurodegenerative and inflammatory diseases, which hinders the development of therapeutic and prophylactic agents for conditions like ALS, FTD, and Huntington's disease.
Innovation Solution
Methods are developed to expand repeat sequences at a target genomic locus in non-human cells using nuclease agents like CRISPR-associated proteins and guide RNAs, introducing double-strand or single-strand breaks near the repeat expansion sequences to increase the number of repeat copies, creating models with expanded hexanucleotide repeat expansions at the C9ORF72 locus.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional animal models are used, then existing therapeutic development can proceed, but the models fail to accurately recapitulate repeat expansion diseases
Solution Approach 1:
The patent introduces repeat expansion sequences into animal genomes before studying disease mechanisms, using CRISPR/Cas9 technology to pre-establish the genetic conditions that mimic human repeat expansion diseases. This preliminary genetic modification enables accurate disease modeling in animals.
Solution Approach 2:
The patent uses CRISPR/Cas9 technology as an intermediary tool to transfer repeat expansion sequences from human genetics into animal genomes. The guide RNA and Cas9 protein act as mediators to precisely insert the pathogenic repeat sequences at specific genomic locations, creating accurate disease models.
2Reliability
If repeat expansion sequences are introduced into animal genomes, then accurate disease models can be created, but the complexity of genetic engineering increases
Solution Approach 1:
The patent employs CRISPR/Cas9 components (guide RNA, Cas9 protein) as intermediaries to simplify the complex process of genetic modification. These intermediaries enable precise targeting and insertion of repeat sequences through programmable RNA-guided nucleases, reducing the overall engineering complexity compared to traditional methods.
Solution Approach 2:
The patent copies repeat expansion sequences from human disease-associated genes into animal genome templates. By replicating the specific repeat sequences (e.g., C9ORF72 hexanucleotide repeats) at corresponding genomic loci in animals, the method creates faithful models without requiring complete genome transplantation.
3Quantity of substance
If CRISPR/Cas9 is used to expand repeat sequences, then the number of repeat copies increases, but off-target effects and genomic instability may occur
Solution Approach 1:
The patent applies CRISPR/Cas9 editing at specific local genomic regions where repeat expansion sequences are already present or precisely targeted. By confining the nuclease activity to defined loci with high sequence homology, the method increases repeat copies locally while minimizing off-target effects in other genomic regions.
Solution Approach 2:
The patent utilizes the potential harmful off-target cutting activity of Cas9 by providing repair templates with desired repeat expansions. The cellular DNA repair machinery, normally responsible for fixing breaks, is redirected to incorporate the beneficial repeat expansion sequence, converting the harmful double-strand break into a useful gene editing outcome.
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
These methods enable the creation of non-human animal models with expanded repeat sequences, allowing for the assessment of therapeutic candidates and providing valuable tools for understanding and treating neurodegenerative disorders associated with repeat expansions.
Implementation Method 1
introducing into a population of cells comprising the repeat expansion sequence a nuclease agent or a nucleic acid encoding the nuclease agent, wherein the nuclease agent cleaves a nuclease target site near the 5′ end or the 3′ end of the repeat expansion sequence
Data Source
AI summary
Nuclease-mediated methods for expanding repeats already present at a genomic locus are provided. Non-human animal genomes, non-human animal cells, and non-human animals comprising a heterologous hexanucleotide repeat expansion sequence inserted at an endogenous C9orf72 locus and methods of making such non-human animal cells and non-human animals through nuclease-mediated repeat expansion are also provided. Methods of using the non-human animal cells or non-human animals to identify therapeutic candidates that may be used to prevent, delay or treat one or more neurodegenerative disorders associated with repeat expansion at the C9orf72 locus are also provided.