CRISPR Transferrin Locus Targeting for Stable Gene Integration

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Solution Overview

Problem

Current gene therapy methods for treating hemophilia A, such as those using Adeno Associated Virus (AAV), are limited by the lack of integration of the therapeutic gene into the host cell genome, leading to transient expression and potential degradation of the therapeutic DNA, especially in children whose livers have not yet reached adult size.

Innovation Solution

A system comprising a DNA endonuclease, a guide RNA (gRNA) with a spacer sequence complementary to the transferrin locus, and a donor template encoding a protein-of-interest (POI) or its functional derivative, which allows for targeted integration and expression of the POI in cells, particularly hepatocytes, using CRISPR/Cas systems.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Duration of action of moving object

If AAV-based gene therapy is used to deliver therapeutic gene, then gene delivery to hepatocytes is achieved, but the therapeutic gene is not integrated into the host genome leading to transient expression

Engineering Contradiction:

Improveduration of therapeutic gene expressionVSAvoidstability of therapeutic gene maintenance

Core Design Contradiction:

Duration of action of moving objectVSReliability

Solution Approach 1:

The CRISPR/Cas system is activated before or during the introduction of the therapeutic gene to create target sites and facilitate integration. The guide RNA and Cas9 nuclease are delivered to cells in advance to prepare the genomic landscape for stable integration of the therapeutic transgene, ensuring permanent incorporation rather than transient episomal maintenance

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The AAV vector serves as an intermediary that delivers both the therapeutic gene and the CRISPR/Cas components. The AAV capsid mediates entry into hepatocytes, while the packaged genetic material includes elements that enable subsequent integration events, bridging the gap between efficient delivery and stable genomic incorporation

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If AAV-based gene therapy is used, then gene delivery is achieved, but the therapeutic DNA is subject to degradation over time

Engineering Contradiction:

Improveprotection of therapeutic DNAVSAvoidpersistence of therapeutic gene

Core Design Contradiction:

ReliabilityVSDuration of action of moving object

Solution Approach 1:

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The host cell's own DNA repair mechanisms are harnessed to integrate the therapeutic gene into the genome. By creating double-strand breaks with Cas9 and providing homology arms on the AAV payload, the cell's natural homology-directed repair pathway is activated to perform the integration, making the process self-sustaining and permanent

Inventive Principle:

Principle #25Self-service

3Adaptability or versatility

If AAV-based gene therapy is used, then treatment is achieved in adult livers, but the therapy is not effective in children whose livers have not reached adult size

Engineering Contradiction:

Improveapplicability across different age groupsVSAvoideffectiveness of gene therapy

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The CRISPR/Cas system with homology-directed repair creates a universal mechanism for gene integration that functions independently of liver size or developmental stage. The same AAV-CRISPR-therapeutic gene construct can be applied to both pediatric and adult patients, making the therapy universally applicable across different age groups and liver sizes

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 enables persistent and stable expression of therapeutic proteins, such as Factor VIII, at levels significantly higher than those achieved with AAV-based therapies, potentially offering a more effective and long-lasting treatment for hemophilia A.

Implementation Method 1

a guide RNA (gRNA) with a spacer sequence complementary to the transferrin locus

Methodology Applied

Scientific EffectComplementary base pairing:

Implementation Method 2

Each of nuclease type is capable of inducing a DNA double-stranded break (DSB) at specific DNA loci, thus triggering two DNA repair pathways

Methodology Applied

Scientific EffectDNA double-stranded break:

Implementation Method 3

the homology-directed repair (HDR) pathway repairs the DSB with the genetic information carried on a donor template

Methodology Applied

Scientific EffectHomology-directed repair:

Data Source

PatentUS12215320B2Compositions and methods for gene editing by targeting transferrin

Publication Date: 2025.02.04 CRISPR THERAPEUTICS AG

AI summary

Provided include compositions, methods, and systems for modulating the expression, function, and/or activity of a target gene, for example a blood-clotting protein such as Factor VIII (FVIII), in a cell by genome editing. Also provided include compositions, methods, and systems for treating a subject having or suspected of having a disorder or health condition, e.g., Hemophilia A, employing ex vivo and/or in vivo genome editing.