CRISPR-Mediated CAR T Cell Integration for Solid Tumors
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Solution Overview
Problem
Current methods for generating modified immune cells, such as CAR-T cells, face challenges including T cell exhaustion, inefficient manufacturing processes, and random gene integration, which affect the efficacy and safety of cancer immunotherapy, particularly in treating solid tumors.
Innovation Solution
A method involving CRISPR/Cas9-mediated insertion of an antigen recognition moiety into specific sites of endogenous cell receptor genes in immune cells, allowing for targeted antigen recognition without viral vectors, enabling efficient and homogeneous production of modified immune cells capable of recognizing tumor-associated antigens.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If viral vectors (retroviral or lentiviral) are used to transduce CAR constructs into T cells, then the T cells can be generated to recognize target antigens, but the gene integration is random which creates safety risks including potential oncogenic transformation and variation in transgene expression
Solution Approach 1:
The patent applies preliminary action by using CRISPR/Cas9 to pre-establish specific integration sites in the T cell genome before introducing the CAR construct. The Cas9 nuclease creates targeted double-strand breaks at predetermined locations (such as the TRAC locus), ensuring that subsequent CAR gene integration occurs at controlled sites rather than randomly, thereby eliminating oncogenic risks while maintaining manufacturing precision
Solution Approach 2:
The patent uses the CRISPR/Cas9 system as an intermediary mechanism to mediate precise gene integration. The guide RNA (gRNA) acts as a specific intermediary that directs the Cas9 nuclease to exact genomic locations, serving as a molecular mediator between the CAR construct and the T cell genome, enabling site-specific integration without the randomness associated with traditional viral transduction
2Quantity of substance
If constitutive promoters (EF1α, CMV, PGK) are used to drive CAR expression, then strong CAR expression is achieved, but T cell exhaustion occurs due to non-natural antigenic stimulation and over-stimulation
Solution Approach 1:
The patent applies parameter changes by replacing constitutive promoters with inducible or regulated promoter systems that allow dynamic control of CAR expression levels. This enables the T cells to modulate their CAR expression in response to environmental cues or treatment protocols, preventing chronic over-stimulation and exhaustion while maintaining high expression when needed, thus improving both persistence and function
Solution Approach 2:
The patent introduces dynamics into the CAR expression system by implementing regulatable promoter elements that allow the expression level to change over time and in response to specific stimuli. This dynamic control mechanism enables the T cells to adapt their CAR expression levels, avoiding the static high-expression state that leads to exhaustion, thereby maintaining long-term functionality and persistence
3Reliability
If T cell exhaustion is addressed by modifying activation mechanisms, then persistence may improve, but the manufacturing process becomes more complex and less efficient
Solution Approach 1:
The patent applies the taking out principle by extracting and separating the CAR expression control from complex regulatory networks. By using well-defined inducible promoter systems with specific induction mechanisms (such as small molecule inducers or specific cytokine responses), the patent simplifies the control architecture while maintaining persistence, allowing straightforward manufacturing protocols without requiring complex multi-component regulatory systems
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 achieves high transgene expression efficiency (20-40%) in immune cells, reduces manufacturing costs and time, and enhances the persistence and function of modified immune cells, improving their ability to target and kill tumor cells.
Implementation Method 1
inserting a nucleic acid sequence encoding an antigen recognition moiety for the target antigen into an endogenous cell receptor gene in an immune cell to form a modified cell receptor gene
Data Source
AI summary
This disclosure relates to immune cells (such as T cells or NK cells) modified in their cell surface receptors to recognize one or more target antigens, in particular tumor-associated antigens. This disclosure also relates to a simple method for editing cell receptors, in particular cell surface receptors naturally expressed by immune cells such as T cells or NK cells, to create modified immune cells (e.g., cytotoxic cells) targeted against one or more target antigens, in particular tumor-associated antigens. Further, this disclosure relates to stem cells modified in one or more endogenous genes encoding one or more cell surface receptors and capable of differentiating into immune cells expressing modified cell surface receptors that recognize one or more target antigens. In addition, this disclosure relates to methods of making such modified stem cells.