Platinum TALEN Editing for TCR Gene Replacement
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Solution Overview
Problem
Current methods for introducing antigen-specific T cell receptors (TCRs) into regulatory T cells (Tregs) face challenges in preventing co-expression with endogenous TCRs, leading to reduced efficacy and specificity.
Innovation Solution
The use of genome editing enzymes, specifically modified TALENs like Platinum TALEN, to edit and remove endogenous TCR genes in Tregs, allowing for the introduction of new TCRs with high specificity and reduced off-target modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional methods are used to introduce exogenous TCR into Tregs, then TCR gene introduction can be achieved, but co-expression with endogenous TCR cannot be completely avoided
Solution Approach 1:
The patent applies preliminary action by using modified TALEN to delete the endogenous TCR gene before introducing the exogenous TCR gene. This pre-deletion ensures that the Treg cells will not co-express endogenous TCR with the introduced exogenous TCR, thereby achieving complete antigen specificity. The sequential approach of first removing endogenous TCR then introducing exogenous TCR resolves the contradiction by eliminating the source of co-expression interference.
2Reliability
If modified TALEN is used to delete endogenous TCR gene, then TCR expression specificity is improved, but device complexity increases
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the TALEN DNA-binding domain to enhance its recognition specificity for the TCR gene sequence. By optimizing specific amino acid residues in the TALEN structure, the system achieves high-fidelity targeting of endogenous TCR genes while minimizing off-target effects. This parameter optimization allows the use of modified TALEN technology without requiring overly complex additional components, as the enhanced specificity is achieved through rational protein design rather than system complexity.
3Productivity
If high cleavage efficiency is achieved with modified TALEN, then endogenous TCR suppression is improved, but off-target gene modification risk may increase
Solution Approach 1:
The patent applies local quality by designing the TALEN system to have highly specific local recognition sequences that match only the intended target site in the endogenous TCR gene. The DNA-binding domain is engineered with precise amino acid sequences that recognize and bind exclusively to the specific genomic locus of the endogenous TCR gene. This localized specificity ensures that high cleavage efficiency is achieved at the target site while minimizing off-target effects, as the TALEN molecules are structurally optimized to distinguish the target sequence from other genomic regions.
Data Source
AI summary
The present disclosure provides a technique whereby the influence of an endogenous TCR is eliminated in TCR gene transfer. A TCR gene is edited using a genome editing enzyme, said genome editing enzyme having one characteristic that amino acids at two specific positions in DNA-binding modules contained in a DNA-binding domain thereof show repeating patterns which differ from one module to another among the four DNA-binding modules. Thus, a lowering in the expression efficiency of the transferred TCR caused by mispairing with an endogenous TCR and the occurrence of a self-reactive TCR are avoided.