Endogenous Cell Surface Protein Editing for Functional T Cell Engineering
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Solution Overview
Problem
Current gene editing techniques for therapeutic use in T cells are limited to correcting single mutations or integrating synthetic genes, lacking flexibility for endogenous modifications, which restricts their applicability in adoptive cellular therapies.
Innovation Solution
The method involves modifying endogenous genes in human T cells by inserting a heterologous nucleic acid sequence encoding a functional domain into a target region, allowing for the generation of T cells with altered functionality and co-regulation of heterologous proteins using targeted nucleases and homologous recombination, enhancing signaling and binding activities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If current gene editing techniques are used to correct single mutations or integrate synthetic genes, then therapeutic applicability is limited to specific conditions, but the complexity of creating new therapeutically useful synthetic DNA sequences is high
Solution Approach 1:
The patent segments the gene editing approach into two distinct strategies: (1) correction of existing mutations at endogenous loci, and (2) integration of synthetic genes at pseudogenes or inactive loci. This segmentation allows each strategy to be optimized independently, expanding therapeutic applicability without proportionally increasing overall complexity
Solution Approach 2:
The patent introduces pseudogenes and inactive genomic loci as intermediary sites for synthetic gene integration. These intermediary elements serve as safe harbors that accept synthetic genes without disrupting essential cellular functions, thereby reducing the complexity and risk associated with creating therapeutically useful synthetic DNA sequences
2Adaptability or versatility
If endogenous genes are modified by inserting heterologous nucleic acid sequences, then T cells with altered functionality are generated, but the risk of off-target effects and side effects increases
Solution Approach 1:
The patent applies local quality by making modifications at specific, predetermined genomic loci rather than random integration. By targeting endogenous genes and pseudogenes with defined characteristics, the modification location is optimized to achieve desired T cell functionality while minimizing disruption to other cellular processes, thereby reducing side effects
Solution Approach 2:
The patent employs prior cushioning by using pseudogenes and inactive loci as buffer zones for synthetic gene integration. These pre-identified safe harbors act as cushions that absorb potential harmful effects of gene integration, protecting essential cellular functions from disruption while still allowing generation of T cells with altered functionality
3Reliability
If targeted nucleases and homologous recombination are used to modify endogenous proteins, then signaling activity is enhanced, but the manufacturing process complexity increases
Solution Approach 1:
The patent employs self-service by utilizing the cell's own homologous recombination machinery to perform the gene modification. Rather than requiring complex external assembly and integration systems, the introduced DNA templates leverage the cell's endogenous repair mechanisms to achieve precise integration, thereby enhancing signaling activity without proportionally increasing manufacturing complexity
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 enables the production of T cells with enhanced functionalities while minimizing side effects, facilitating immune response enhancement in human subjects through targeted antigen-specific TCR complexes.
Implementation Method 1
a targeted nuclease that cleaves a target region in a nucleic acid encoding the endogenous cell surface protein to create an insertion site in the genome of the cell
Implementation Method 2
allowing homologous recombination to take place, thereby inserting the nucleic acid sequence in the insertion site
Data Source
AI summary
Provided herein are methods and compositions for modifying an endogenous cell surface protein in a human cell by inserting a heterologous nucleic acid sequence in a target region of a nucleic acid encoding the endogenous cell surface protein.