CAR T-Cell Gene Editing for Targeted Integration and TCR Knockout

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Solution Overview

Problem

Current gene therapy methods face challenges such as unpredictable transgene integration, potential oncogene activation, and immune response issues in adoptive cell therapy, limiting the efficiency and specificity of engineered T cells for targeted treatments.

Innovation Solution

Introduce a chimeric antigen receptor (CAR) into T cells with targeted integration into TCR and B2M genes, and modify the cells to lack a functional endogenous TCR and HLA complex, optionally expressing modified HLA-E or HLA-G, to enhance specificity and durability of antigen recognition.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Duration of action of stationary object

If random integration of transgene is used, then durable expression is achieved, but oncogene activation risk increases

Engineering Contradiction:

Improvetransgene expression durabilityVSAvoidoncogene activation risk

Core Design Contradiction:

Duration of action of stationary objectVSObject-affected harmful factors

Solution Approach 1:

The patent uses site-specific nucleases (ZFNs, TALENs, or CRISPR/Cas systems) as intermediary tools to mediate the integration process. These nucleases create targeted double-strand breaks at predetermined safe harbor loci, enabling controlled transgene insertion that avoids random integration risks while maintaining durable expression through genomic integration.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The invention changes the integration parameter from random to site-specific by introducing nuclease-mediated targeting. This parameter change allows the transgene to be inserted at predetermined safe harbor loci with specific genomic coordinates, transforming the integration process from unpredictable to precisely controlled, thereby eliminating oncogene activation risk while preserving expression durability.

Inventive Principle:

Principle #35Parameter changes

2Manufacturing precision

If site-specific nuclease integration is used, then transgene positioning precision is improved, but integration efficiency decreases

Engineering Contradiction:

Improvetransgene integration precisionVSAvoidintegration efficiency

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The patent employs preliminary action by first introducing the site-specific nuclease to create a double-strand break at the target locus before introducing the transgene donor. This pre-prepared genomic break facilitates subsequent transgene insertion, improving integration efficiency at the precise target site without compromising positioning accuracy. The homology-directed repair mechanism is activated in advance to enable efficient repair and integration.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The invention uses a composite approach combining multiple molecular components: site-specific nucleases (ZFNs, TALENs, or CRISPR/Cas systems) for precise targeting, homology arms from donor DNA for directed integration, and cellular repair machinery for final incorporation. This composite system achieves both high precision and improved efficiency by leveraging multiple molecular mechanisms working together.

Inventive Principle:

Principle #40Composite materials

3Reliability

If endogenous TCR is retained, then immune response capability is maintained, but CAR specificity is reduced

Engineering Contradiction:

Improveimmune response capabilityVSAvoidantigen recognition specificity

Core Design Contradiction:

ReliabilityVSManufacturing precision

Solution Approach 1:

The patent applies the taking out principle by removing or inactivating the endogenous TCR alpha or beta chain genes through targeted nuclease cleavage. This extraction eliminates the competing antigen recognition pathway, ensuring that the engineered CAR becomes the sole functional antigen recognition molecule on the T cell surface, thereby maximizing CAR specificity while maintaining immune response capability through the CAR itself.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

Instead of attempting to enhance endogenous TCR specificity, the invention inverts the approach by eliminating TCR function entirely and replacing it with the engineered CAR system. This inversion allows the CAR to dominate antigen recognition without competition from endogenous TCR, achieving superior specificity for the desired target antigen while maintaining T cell immune functionality through the adopted CAR mechanism.

Inventive Principle:

Principle #13The other way round (Inversion)

Data Source

PatentUS12516098B2Chimeric antigen receptor mediated cell targeting

Publication Date: 2026.01.06 SANGAMO THERAPEUTICS INC

AI summary

Disclosed herein are methods and compositions for engineering cells to express a CAR where the cells also have inactivated TCR, HPRT, PD1, CISH and/or HLA genes, using engineered nucleases comprising at least one DNA binding domain and a cleavage domain or cleavage half-domain in conditions able to preserve cell viability. Polynucleotides encoding nucleases, vectors comprising polynucleotides encoding nucleases and cells comprising polynucleotides encoding nucleases and/or cells comprising nucleases are also provided.