dgRNA Activation Screening for CD8+ T Cell Effector Boost
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Solution Overview
Problem
Existing genetic screens in cancer immunology are limited to loss-of-function approaches, and gain-of-function screens to boost CD8+ T cell anti-tumor function have not been explored due to technical challenges in manipulating primary T lymphocytes.
Innovation Solution
A vector comprising a first promoter, a dgRNA expression cassette, a MS2-loop-containing chimeric backbone, and a cassette expressing a fusion protein of Thy 1.1 and one or more transcription activators, along with a non-naturally occurring dgRNA library, is used to enhance CD8+ T cell effector function through high-throughput activation screening.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If loss-of-function screens using RNAi or CRISPR/Cas9 are used to identify key regulators, then T cell differentiation and cancer immunotherapy modulation can be identified, but gain-of-function targets to augment anti-tumor function cannot be identified
Solution Approach 1:
The patent inverts the conventional loss-of-function screening approach by implementing a gain-of-function screen using CRISPR/Cas9 activation. Instead of knocking down genes to observe reduced function, the system uses guide RNAs to activate gene expression and observe enhanced T cell effector function, thereby identifying genes that boost anti-tumor activity when overexpressed.
Solution Approach 2:
The patent changes the functional parameter being measured from loss of function to gain of function. By using CRISPR/Cas9 activation instead of RNAi-mediated knockdown, the system measures increases in T cell effector function (such as degranulation, cytokine production, and cytotoxicity) resulting from gene activation, enabling identification of genes that enhance anti-tumor immunity.
2Loss of information
If primary T lymphocytes are manipulated for genetic screens, then gain-of-function targets can be identified, but technical challenges in genetic manipulation arise
Solution Approach 1:
The patent replaces complex viral-based genetic manipulation methods with a CRISPR/Cas9 activation system that can be delivered through simpler means. The guide RNA and Cas9 protein are introduced together, eliminating the need for complex viral vectors and enabling efficient gene activation in primary T cells without requiring extensive genetic engineering infrastructure.
Solution Approach 2:
The patent uses guide RNAs as intermediaries to direct Cas9 protein to specific gene loci. This intermediary mechanism enables precise gene activation without requiring direct manipulation of the genome through viral integration or transfection, simplifying the overall process while maintaining high specificity and efficiency in primary T cells.
3Productivity
If high-throughput screening is implemented to identify genetic targets, then effector function enhancement can be screened systematically, but screening complexity increases
Solution Approach 1:
The patent segments the screening system into modular components: a library of guide RNAs targeting individual genes, a CRISPR/Cas9 activation mechanism, and readout assays for effector function measurement. This segmentation enables parallel processing of multiple genes simultaneously while maintaining manageable complexity through standardized reagents and protocols.
Solution Approach 2:
The patent creates a universal CRISPR/Cas9 activation platform that can be applied across multiple gene targets and T cell types using the same core reagents and methodology. The guide RNA library serves as a universal tool that can screen for activation of any gene in the human genome, enabling high-throughput identification of effector function enhancers without requiring target-specific reagents.
Data Source
AI summary
The present invention includes dead-guide RNA (dgRNA) libraries and methods of use in immunology and immunotherapy thereof. Also provided are engineered primary or chimeric antigen receptor (CAR) T cells that overexpress Prodh2, Ccnblip1, Sreklip1, or Wdr37 or a fragment thereof, and methods of use in immunology and immunotherapy thereof.