Multiplex Immune Cell Editing for Suppression-Resistant CAR Therapy
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Solution Overview
Problem
Current immunotherapeutic approaches for cancer treatment, particularly against solid tumors, are limited due to reduced tumor antigen expression, expression of inhibitory receptors, and induction of suppressive cells in the microenvironment that hinder immune response.
Innovation Solution
Multiplex editing of immune cells using CRISPR/Cas9 technology to disrupt multiple genes associated with immune suppression, such as NKG2A, CISH, and TGFBR2, and introduce heterologous proteins like CARs at specific gene loci to enhance cytotoxicity and persistence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional immunotherapeutic approaches are used, then treatment simplicity is maintained, but antitumor efficacy is limited due to inhibitory receptors and suppressive microenvironment
Solution Approach 1:
The patent segments the immune cell modification process into multiple targeted gene disruptions (NKG2A, CISH, TGFBR2, etc.) and heterologous protein introductions (CARs, TCRs), allowing systematic addressing of different suppressive mechanisms separately and then integrating their effects for enhanced overall antitumor efficacy
Solution Approach 2:
The patent combines multiple gene editing operations and protein introductions into a single immune cell therapy product, merging the effects of inhibitory receptor blockade, suppressive microenvironment targeting, and heterologous antigen recognition to achieve synergistic antitumor activity that exceeds individual approaches
2Reliability
If multiple genes are disrupted simultaneously, then immune cell functionality is enhanced, but manufacturing complexity increases
Solution Approach 1:
The patent employs pre-designed guide RNAs and editing templates that are prepared in advance, allowing multiple gene disruptions to be performed in a systematic sequence. The guide RNAs are pre-synthesized and the editing machinery is pre-assembled, streamlining the manufacturing process despite the multiple targets
Solution Approach 2:
The patent utilizes CRISPR-Cas9 technology to achieve precise parameter changes in gene expression (from normal expression to complete disruption or partial knockdown), allowing controlled modification of multiple genes with high precision and reproducibility, thereby managing manufacturing complexity through standardized editing protocols
3Measurement precision
If heterologous proteins are introduced at genomic loci, then target specificity is improved, but risk of off-target effects increases
Solution Approach 1:
The patent introduces heterologous proteins (CARs, TCRs) at specific genomic loci within the immune cell genome, creating local areas of high target recognition precision. The proteins are inserted at defined locations to ensure proper expression and function while minimizing disruption to other genomic regions, thereby reducing off-target effects
Solution Approach 2:
The patent uses guide RNAs as intermediary molecules that mediate the introduction of heterologous proteins at specific genomic loci. The guide RNAs provide sequence-specific targeting, acting as intermediaries between the editing machinery and the genomic DNA, ensuring precise localization and minimizing off-target effects through careful guide RNA design
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
Enhances antitumor cytotoxicity, in vivo proliferation, and immune cell function by reducing suppressive effects, allowing immune cells to better target and eliminate tumor cells.
Implementation Method 1
Multiplex editing of immune cells using CRISPR/Cas9 technology to disrupt multiple genes associated with immune suppression
Implementation Method 2
introduce heterologous proteins like CARs at specific gene loci to enhance cytotoxicity and persistence
Implementation Method 3
enhances antitumor cytotoxicity, in vivo proliferation, and immune cell function
Data Source
AI summary
Provided herein are methods for producing immune cells with disruption of multiple genes. Further provided are methods for inserting a chimeric antigen receptor at a gene locus of an immune cell.