Engineered Meganucleases for TCR Alpha Gene-Specific Cleavage
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Solution Overview
Problem
Current CAR T cell therapies for cancer treatment are limited by the expression of the endogenous T cell receptor, which can lead to graft-versus-host-disease (GVHD) when administered to allogeneic patients, and the production of autologous CAR T cells is time-consuming and expensive.
Innovation Solution
Development of engineered meganucleases that specifically target and disrupt the TCR alpha constant region gene, allowing for the generation of allogeneic CAR T cells with reduced endogenous T cell receptor expression, using optimized TRC 1-2 meganucleases with improved specificity and reduced off-target cutting, and enhanced efficiency in modifying the TCR alpha constant region gene.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If autologous CAR T cells are produced to ensure immune tolerance and prevent GVHD, then safety is improved, but production time and cost increase significantly
Solution Approach 1:
The patent applies preliminary action by pre-modifying T cells from healthy donors ex vivo to eliminate endogenous TCR expression and introduce CAR receptors before clinical need arises. These pre-prepared allogeneic CAR T cells can be stored and rapidly administered when needed, eliminating the time-consuming process of collecting, modifying, and expanding patient-specific T cells while maintaining safety through the absence of endogenous TCR that causes GVHD
Solution Approach 2:
The patent uses copying by creating standardized allogeneic CAR T cell products from healthy donor T cells that can be replicated and stored. Instead of creating unique autologous products for each patient, the modified donor T cells serve as a template that can be expanded and administered to multiple patients, significantly reducing production time while maintaining therapeutic efficacy
2Object-generated harmful factors
If engineered meganucleases are used to disrupt TCR alpha constant region gene, then endogenous T cell receptor expression is reduced preventing GVHD, but off-target cutting may occur causing harmful effects
Solution Approach 1:
The patent applies parameter changes by systematically optimizing multiple parameters of the engineered meganuclease including recognition sequence specificity, catalytic activity, and binding affinity. The meganuclease was engineered with precise amino acid substitutions in the recognition domain to enhance specificity for the target TCR alpha constant region gene while minimizing off-target effects. Expression levels and timing were also optimized to achieve complete TCR knockout without excessive nuclease activity that could cause off-target damage
Solution Approach 2:
The patent implements feedback by using high-throughput sequencing and genomic analysis to identify and characterize off-target cutting events. This information feeds back into further optimization of the meganuclease design, allowing iterative improvement of specificity. The feedback mechanism enables continuous refinement of the enzyme's recognition properties to eliminate harmful off-target effects while maintaining effective disruption of the target gene
3Productivity
If third-party T cells are used to create off-the-shelf CAR T cells, then production time is reduced and availability is improved, but immune tolerance is lost increasing GVHD risk
Solution Approach 1:
The patent applies taking out by specifically removing the problematic endogenous TCR alpha constant region gene from donor T cells while preserving the desired CAR receptor and other functional elements. By selectively eliminating only the TCR component responsible for GVHD through meganuclease-mediated disruption, the patent retains the productivity benefits of allogeneic cells while extracting the harmful GVHD-inducing capability
Solution Approach 2:
The patent converts the potential harm of allogeneic T cell rejection and GVHD into a benefit by deliberately engineering the donor T cells to lack endogenous TCR expression. This conversion transforms what would normally be a harmful immune response into a safe, universal product that can be administered to any patient without risk of GVHD, while maintaining the productivity advantages of off-the-shelf availability
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 engineered meganucleases enable the production of allogeneic CAR T cells with reduced GVHD risk, improved efficiency, and enhanced T cell expansion, overcoming the limitations of autologous CAR T cell production and time constraints.
Implementation Method 1
engineered meganucleases that specifically target and disrupt the TCR alpha constant region gene, allowing for the generation of allogeneic CAR T cells with reduced endogenous T cell receptor expression, using optimized TRC 1-2 meganucleases with improved specificity
Implementation Method 2
engineered meganucleases that specifically target and disrupt the TCR alpha constant region gene
Data Source
AI summary
The present invention encompasses engineered nucleases which recognize and cleave a recognition sequence within the first exon of the human T cell receptor (TCR) alpha constant region gene. The engineered meganucleases can exhibit at least one optimized characteristic, such as enhanced (i.e., increased) specificity or efficiency of cleavage, when compared to the first-generation meganuclease TRC 1-2x.87EE. The present invention also encompasses methods of using such engineered nucleases to make genetically-modified cells, and the use of such cells in a pharmaceutical composition and in methods for treating diseases, such as cancer.