CRISPR-Cas9 CAR T Cells Targeting uPAR Senescent Cells
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Solution Overview
Problem
Current senolytic therapies for eliminating senescent cells, such as those targeting urokinase Plasminogen Activator Receptor (uPAR), rely on retroviral vectors and lack the potency and specificity provided by CRISPR-Cas9 genome editing, which is essential for effective treatment of senescence-associated diseases like neurodegenerative disorders.
Innovation Solution
Development of a virus-free method using CRISPR/Cas9 and homology-directed repair (HDR) to integrate a chimeric antigen receptor (CAR) gene into T cells, enabling precise genomic integration of a CAR transgene that targets uPAR, thereby generating potent CAR T cells capable of eliminating senescent cells without viral vectors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If retroviral vectors are used to deliver CAR transgene to T cells, then T cells can be engineered to target senescent cells, but the therapy lacks the potency and specificity provided by CRISPR-Cas9 genome editing
Solution Approach 1:
The patent divides the genome editing process into distinct functional modules: CRISPR-Cas9 nuclease for targeted DNA cleavage, homology-directed repair template for precise transgene integration, and selection markers for identifying successfully edited cells. This segmentation allows each component to be optimized independently while maintaining overall system effectiveness.
Solution Approach 2:
The patent uses homology-directed repair templates as intermediaries to mediate the integration of CAR transgenes into specific genomic loci. These templates serve as the bridge between the CRISPR-Cas9 cleavage activity and the desired transgene insertion, enabling precise and controlled genome modification without direct viral integration.
2Manufacturing precision
If CRISPR-Cas9 genome editing is used to integrate CAR transgene, then potency and specificity are enhanced, but the manufacturing process becomes more complex
Solution Approach 1:
The patent prepares homology-directed repair templates in advance with all necessary genomic sequences and flanking regions required for precise transgene integration. This preliminary preparation of integration templates streamlines the actual genome editing process by providing ready-to-use DNA constructs that direct CRISPR-Cas9 to the correct genomic loci, reducing manufacturing complexity.
Solution Approach 2:
The patent optimizes multiple parameters of the CRISPR-Cas9 system including guide RNA design, Cas9 nuclease concentration, homology arm length of integration templates, and electroporation conditions. By systematically adjusting these parameters, the patent achieves high transgene integration precision while maintaining manufacturing feasibility through standardized protocols.
3Productivity
If viral vectors are used for CAR T cell production, then transgene delivery is efficient, but the therapy lacks the specificity and precision of CRISPR-mediated genome editing
Solution Approach 1:
The patent extracts the transgene delivery function from viral vectors and replaces it with CRISPR-Cas9 mediated homology-directed repair. This extraction eliminates the need for viral integration while maintaining efficient transgene delivery through precisely designed genomic integration loci that ensure high expression levels and stable inheritance.
Solution Approach 2:
The patent uses homology-directed repair templates as precise copies of the desired genomic integration site. These templates contain exact sequences matching the target locus flanking regions, enabling accurate copying of the integration site structure and ensuring faithful reconstruction of the genomic locus with the inserted CAR transgene, thereby achieving high specificity.
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 approach results in high-efficiency, specific elimination of senescent cells in vitro and in vivo, demonstrating potential for treating neurodegenerative diseases like Alzheimer's and Parkinson's by enhancing the therapeutic efficacy of CAR T cell therapies.
Implementation Method 1
introducing into a population of unmodified T cells a Cas9 ribonucleoprotein (RNP) and the HDR template to provide the CAR T cells
Implementation Method 2
Development of a virus-free method using CRISPR/Cas9 and homology-directed repair (HDR) to integrate a chimeric antigen receptor (CAR) gene into T cells
Implementation Method 3
targeting the cell surface antigen urokinase Plasminogen Activator Receptor (uPAR)... a polynucleotide encoding a polypeptide that specifically binds uPAR
Data Source
AI summary
Described herein are methods using CRISPR-Cas9 and DNA templates that can generate chimeric antigen receptors (CARs) on T cells to target the cell surface protein urokinase Plasminogen Activator Receptor (uPAR) on senescent cells. Also described are methods of preparing CAR T cells, their use to treat neurodegenerative disease, stroke, craniocerebral trauma and/or accident, or elderly individuals in need of treatment for aging.