Modified TRAC Intron CAR T Cells for GVHD Reduction
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Solution Overview
Problem
Existing 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
Genetically modify T cells by targeting and inserting a sequence of interest, such as a CAR coding sequence, into the intron 5' upstream of TRAC exon 1 in the T cell receptor alpha gene, using a monomeric engineered meganuclease that preserves the endogenous splice sites, ensuring most TCR− cells become TCR−/CAR+.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CAR T cells are produced using autologous T cells to ensure immune tolerance, then GVHD risk is reduced, 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 create allogeneic CAR T cells that are ready for immediate use. The T cells are genetically modified outside the patient's body to express CAR and have their endogenous TCR expression reduced, allowing them to be prepared in advance and stored for later administration without requiring time-consuming autologous cell collection and expansion
Solution Approach 2:
The patent uses allogeneic T cells from healthy donors as a substitute (copy) for patient-specific autologous T cells. These donor-derived T cells are genetically engineered to mimic the desired therapeutic function while avoiding the time delays associated with autologous cell production. The copied cells are then administered to multiple patients as an off-the-shelf product
2Reliability
If endogenous T cell receptor expression is maintained in CAR T cells, then T cell functionality is preserved, but GVHD occurs when administered to allogeneic patients
Solution Approach 1:
The patent applies the extraction principle by specifically reducing or eliminating the endogenous T cell receptor from the T cell surface while preserving the CAR expression. Using CRISPR/Cas9 technology, the TCR alpha chain gene is targeted to knock down endogenous TCR expression, thereby removing the harmful component (endogenous TCR that causes GVHD) while maintaining the therapeutic component (CAR expression)
Solution Approach 2:
The patent applies local quality by differentiating between two types of receptor expression on the T cell surface: endogenous TCR expression is reduced or eliminated to prevent GVHD, while CAR expression is maintained and enhanced to preserve therapeutic functionality. This selective modification creates a localized quality difference where only the harmful receptor type is removed while the therapeutic receptor type is preserved
3Productivity
If allogeneic T cells are used to reduce production time, then immediate availability is achieved, but GVHD risk increases due to endogenous TCR expression
Solution Approach 1:
The patent converts the potential harm of using allogeneic T cells (which normally would cause GVHD due to endogenous TCR expression) into a benefit by using CRISPR/Cas9 technology to specifically knock down the endogenous TCR. The same allogeneic cells that would have been harmful are transformed into safe, effective therapeutic products by removing the harmful TCR component while preserving the therapeutic CAR component
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 eliminates the need for enrichment of CAR+ cells from mixed populations and reduces the risk of GVHD, enabling the production of allogeneic CAR T cells that are ready for immediate use.
Implementation Method 1
Genetic modification of genomic DNA can be performed using site-specific, rare-cutting endonucleases that are engineered to recognize DNA sequences in the locus of interest
Data Source
AI summary
The present invention provides a genetically-modified T cell comprising in its genome a modified human T cell receptor alpha gene. The modified T cell receptor alpha gene comprises an exogenous sequence of interest inserted into an intron within the T cell receptor alpha gene that is positioned 5′ upstream of TRAC exon 1. The exogenous sequence of interest can comprise an exogenous splice acceptor site and/or a poly A signal, which disrupts expression of the T cell receptor alpha subunit. The sequence of interest can also include a coding sequence for a polypeptide, such as a chimeric antigen receptor. Additionally, the endogenous splice donor site and the endogenous splice acceptor site flanking the intron are unmodified and/or remain functional. The invention further provides compositions and methods for producing the genetically-modified cell, and populations of the cell, and methods for the treatment of a disease, such as cancer, using such cells.