C9orf72 CRISPR Interference for Repeat Transcript Repression

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Solution Overview

Problem

There are no effective cures for amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) caused by C9orf72 gene hexanucleotide repeat expansions, and the physiological function of the C9orf72 protein is not well understood.

Innovation Solution

Utilization of CRISPR/Cas systems, specifically nuclease-inactive Cas proteins and guide RNAs, to target and repress transcription from the C9orf72 exon 1A transcription start site and hexanucleotide repeat expansion sequences, reducing or abolishing the expression of sense and antisense transcripts containing the repeat expansion.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If CRISPR/Cas systems with nuclease-inactive Cas proteins are used to repress transcription, then harmful transcripts are reduced without causing DNA breaks, but the mechanism relies on steric blocking which may be less potent than nuclease activity

Engineering Contradiction:

Improvesafety of CRISPR systemVSAvoidtranscript expression

Core Design Contradiction:

ReliabilityVSObject-generated harmful factors

Solution Approach 1:

The patent extracts the nuclease activity from the CRISPR/Cas system by using nuclease-inactive Cas proteins (dCas9), isolating the transcriptional repression function through steric blocking by guide RNA-bound Cas proteins. This separation eliminates DNA breaking while maintaining transcript suppression capability.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The guide RNA acts as an intermediary molecule that directs the nuclease-inactive Cas protein to specific target sequences. The guide RNA-Cas protein complex serves as a mediator that blocks transcription through steric hindrance without requiring DNA cleavage, enabling safe and specific transcript repression.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Productivity

If guide RNAs target sequences upstream of or proximate to the transcription start site, then transcription initiation is blocked, but off-target effects may occur

Engineering Contradiction:

Improvetranscription repression efficiencyVSAvoidoff-target effects

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent applies local quality by designing guide RNAs with specific 20-nucleotide targeting sequences that are complementary to unique regions upstream of or proximate to the C9orf72 exon 1A transcription start site. This localized targeting ensures high-specificity binding and transcription blocking at the intended location while minimizing off-target effects through sequence specificity.

Inventive Principle:

Principle #3Local quality

3Reliability

If multiple guide RNAs targeting different sequences are used simultaneously, then comprehensive transcript repression is achieved, but system complexity increases

Engineering Contradiction:

Improvecompleteness of transcript suppressionVSAvoidCRISPR system structure

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent segments the transcription repression task into multiple independent guide RNA-Cas protein complexes, each targeting a different sequence location upstream of or proximate to the transcription start site. This segmentation allows simultaneous blocking of multiple transcription initiation sites through parallel action of separate CRISPR complexes, achieving comprehensive suppression without requiring a single complex system.

Inventive Principle:

Principle #1Segmentation

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

Reduces or abolishes the expression of harmful C9orf72 hexanucleotide-repeat-containing transcripts, potentially ameliorating symptoms of ALS and FTLD, providing a therapeutic and prophylactic approach for these diseases.

Implementation Method 1

a first guide RNA comprising a first DNA-targeting segment that targets a first guide RNA target sequence upstream of or proximate to the C9orf72 exon 1A transcription start site, wherein the first CRISPR/Cas complex binds to the first guide RNA target sequence

Methodology Applied

Scientific EffectBase pairing:

Data Source

PatentUS20250381303A1Crispr interference therapeutics for c9ORF72 repeat expansion disease

Publication Date: 2025.12.18 REGENERON PHARMACEUTICALS INC

AI summary

Guide RNAs and CRISPR/Cas systems targeting a C9orf72 gene, lipid nanoparticles or viral vectors comprising such CRISPR/Cas systems, and cells or animals comprising such CRISPR/Cas systems are provided. Methods of repressing transcription from a C9orf72 exon 1 A transcription start site and/or repressing transcription of sense and/or antisense transcripts that comprise the hexanucleotide repeat expansion sequence in a C9orf72 gene using the CRISPR/Cas systems are also provided, as well as use of the CRISPR/Cas systems in prophylactic and therapeutic applications for treatment and/or prevention of a C9orf72 hexanucleotide repeat expansion associated disease and/or for ameliorating at least one symptom associated with such disease.