C9ORF72 Repeat Expansion Excision in iPSCs Using CRISPR/Cas9

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Solution Overview

Problem

Current gene editing approaches for the C9orf72 repeat expansion mutation, which causes frontotemporal dementia and amyotrophic lateral sclerosis, face challenges such as off-target editing, disruption of nearby regulatory regions, and inefficiency in post-mitotic cells, making them unsafe and ineffective for therapeutic intervention.

Innovation Solution

CRISPR-based methods targeting the C9orf72 repeat expansion mutation, including bi-allelic excision of the repeat expansion region, allele-specific excision of the mutant allele, and excision of the regulatory region (exon 1A), which normalize RNA abnormalities and TDP-43 pathology, while minimizing off-target effects.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If CRISPR-based gene editing is used to remove the C9orf72 repeat expansion, then therapeutic effectiveness is improved, but off-target editing risks increase

Engineering Contradiction:

Improvetherapeutic effectivenessVSAvoidoff-target editing

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent applies local quality by designing guide RNAs with varying degrees of complementarity to different target sites. The first guide RNA has higher complementarity to the repeat expansion region for effective cutting, while the second guide RNA has lower complementarity to reduce off-target effects. This differential design allows selective editing at the desired locus while minimizing unintended genomic modifications.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent segments the gene editing process into two distinct steps using two different guide RNAs. The first step uses a guide RNA targeting the repeat expansion region to create an initial break, and the second step uses a different guide RNA targeting a downstream site to create a second break. This segmentation allows controlled editing through NHEJ repair, removing the harmful repeat while preserving the rest of the gene structure.

Inventive Principle:

Principle #1Segmentation

2Reliability

If editing strategies target the repeat expansion region, then pathogenic dipeptide repeats are eliminated, but nearby regulatory regions may be disrupted

Engineering Contradiction:

Improveelimination of pathogenic dipeptide repeatsVSAvoidregulatory region integrity

Core Design Contradiction:

ReliabilityVSManufacturing precision

Solution Approach 1:

The patent extracts only the harmful repeat expansion sequence from the C9orf72 gene while leaving the rest of the gene structure intact. By using CRISPR-Cas9 to create breaks flanking the repeat region and relying on NHEJ to join the surrounding sequences, the method removes the pathogenic element (repeat expansion and associated dipeptide repeats) while preserving the regulatory regions and coding sequence necessary for normal gene function.

Inventive Principle:

Principle #2Taking out (Extraction)

3Manufacturing precision

If homology directed repair is used for gene editing, then editing accuracy is improved, but efficiency in post-mitotic cells decreases

Engineering Contradiction:

Improveediting accuracyVSAvoidediting efficiency

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The patent employs a transient, disposable editing approach using plasmid DNA or RNA delivered temporarily to post-mitotic neurons. The CRISPR components are introduced, perform their editing function, and then degrade naturally. This avoids the need for permanent integration or complex homology-directed repair templates, making the process feasible in non-dividing cells where sustained editing machinery would be required.

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

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 effectively eliminate pathogenic dipeptide repeats and restore normal RNA and protein expression, reducing TDP-43 pathology in patient-derived neurons, providing a promising therapeutic approach for C9orf72-related diseases.

Implementation Method 1

CRISPR gene editing holds promise to cure or arrest monogenic disease, if we know which edit will be curative at the cellular level, and can achieve such an edit reliably, safely and effectively

Methodology Applied

Scientific EffectCRISPR/Cas9 gene editing:

Implementation Method 2

the present invention provides an efficacious and safe CRISPR-based method of editing the C9orf72 repeat expansion mutation, and first in class guide RNAs of use in carrying out this method

Methodology Applied

Scientific EffectNucleic acid hybridization:

Implementation Method 3

editing strategies that utilize homology directed repair are inefficient in post-mitotic cells

Methodology Applied

Scientific EffectNon-homologous end joining:

Data Source

PatentUS20250313819A1Therapeutic crispr/CAS9 gene editing approaches to the c9ORF72 repeat expansion mutation in ipscs

Publication Date: 2025.10.09 RGT UNIV OF CALIFORNIA

AI summary

There are provided in vitro and in vivo methods of editing the C9ORF72 repeat expansion mutation using a nuclease to edit a nucleic acid in which the expansion is found. An exemplary method uses a Cas-9 editing system. Guide nucleic acids for editing the repeat expansion mutation are provided. Also provided is a method of mitigating or eliminating symptoms arising in a subject due to the presence of the mutation in the subject's genome.