CRISPR gRNA Segmentation for Hemoglobinopathy Editing

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Solution Overview

Problem

Current genome editing technologies, such as CRISPR/Cas systems, face challenges in effectively modifying cells to increase fetal hemoglobin expression and decrease beta globin expression for treating hemoglobinopathies like sickle cell disease and beta thalassemia, as they require precise targeting and minimal off-target effects.

Innovation Solution

Development of CRISPR systems, including Cas9 CRISPR systems, that utilize gRNA molecules with specific targeting domains complementary to the BCL11A gene, BCL11a enhancer, or hereditary persistence of fetal hemoglobin (HPFH) regions to modify hematopoietic stem and progenitor cells, increasing fetal hemoglobin expression and decreasing beta globin expression.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If CRISPR/Cas systems are used to modify cells to increase fetal hemoglobin expression, then therapeutic benefit is improved, but precision of targeting and minimal off-target effects must be maintained

Engineering Contradiction:

Improvetherapeutic benefitVSAvoidtargeting precision

Core Design Contradiction:

ReliabilityVSManufacturing precision

Solution Approach 1:

The gRNA is divided into two separate molecules: a crRNA containing the targeting domain complementary to BCL11A gene/enhancer/HPFH region, and a tracrRNA. This segmentation allows independent optimization of targeting specificity (crRNA) and Cas9 binding functionality (tracrRNA), thereby maintaining high targeting precision while achieving therapeutic benefit through increased fetal hemoglobin expression.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The tracrRNA serves as an intermediary molecule that mediates between the crRNA (targeting component) and the Cas9 protein. It facilitates precise binding of the CRISPR system to the target sequence while minimizing off-target effects, thus maintaining targeting precision during the therapeutic modification process.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Productivity

If gRNA molecules with specific targeting domains are used to target BCL11A gene or enhancer regions, then fetal hemoglobin expression is increased, but specificity of targeting must be maintained to avoid off-target effects

Engineering Contradiction:

Improvefetal hemoglobin expressionVSAvoidspecificity of targeting

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The crRNA is designed with a targeting domain that is locally complementary to specific sequences in the BCL11A gene, BCL11a enhancer, or HFPH region. This local sequence complementarity ensures that the CRISPR system acts precisely on the intended target region to modulate fetal hemoglobin expression without affecting other genomic regions, thereby maintaining high specificity while achieving the desired productivity increase.

Inventive Principle:

Principle #3Local quality

3Reliability

If CRISPR systems modify hematopoietic stem and progenitor cells to decrease beta globin expression, then treatment efficacy is improved, but minimal off-target effects must be ensured

Engineering Contradiction:

Improvetreatment efficacyVSAvoidoff-target effects

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The system uses a specifically designed gRNA with a targeting domain that is predetermined to be complementary only to the intended target sequences (BCL11A gene, BCL11a enhancer, or HFPH region). This preliminary design of the targeting sequence ensures that the CRISPR system will only bind and modify the correct target, preventing off-target effects before the editing process begins, while still achieving high treatment efficacy through decreased beta globin expression.

Inventive Principle:

Principle #10Preliminary action

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

The CRISPR systems achieve significant increases in fetal hemoglobin expression and decreases in beta globin expression in targeted cells, potentially treating hemoglobinopathies with high specificity and minimal off-target effects, as demonstrated by increased fetal hemoglobin production in erythroid progeny.

Implementation Method 1

the crRNA includes a targeting domain that is complementary with a target sequence of the BCL11A gene, a BCL11a enhancer, or a HFPH region

Methodology Applied

Scientific EffectComplementary base pairing:

Data Source

PatentUS20240002843A1Compositions and methods for the treatment of hemoglobinopathies

Publication Date: 2024.01.04 NOVARTIS AG

AI summary

The present invention is directed to genome editing systems, reagents and methods for the treatment of hemoglobinopathies.