CRISPR/Cpf1 st-crRNA Editing for Universal CAR T Cells
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Solution Overview
Problem
Current methods for CAR T cell immunotherapy are hindered by the lack of readily available, potent, antigen-specific T lymphocytes, leading to high costs and time consumption, and the risk of graft-versus-host disease due to immune recognition of histocompatibility antigens, with existing gene editing technologies like ZFN and TALEN being inefficient in preventing these issues.
Innovation Solution
The use of exogenous nucleic acids encoding stem-loop CRISPR RNA (st-crRNA) and the Cpf1 enzyme for efficient gene editing in T cells, specifically targeting the TCR alpha chain, TCR beta chain, and beta-2 microglobulin to generate universal CAR T cells, reducing the risk of graft-versus-host disease and improving the efficiency of genome engineering.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR/Cas9 technology is used for gene editing in T cells, then genome engineering capability is improved, but editing efficiency and reliability remain insufficient for therapeutic applications
Solution Approach 1:
The patent changes the key parameter of the CRISPR system by replacing Cas9 with Cpf1 enzyme, which has different PAM recognition requirements and cutting mechanisms. This parameter change enables more efficient and reliable gene editing in primary human T cells, achieving up to 80% disruption of TCR expression while reducing off-target effects compared to conventional CRISPR/Cas9 approaches
2Reliability
If autologous CAR T cells are manufactured with patient-specific modifications, then therapeutic efficacy is improved, but manufacturing time and cost increase
Solution Approach 1:
The patent creates universal CAR T cells by disrupting TCR alpha chain, TCR beta chain, and beta-2 microglobulin genes to eliminate HLA restriction. These universal T cells can be manufactured once and used across multiple patients, eliminating the need for patient-specific customization while maintaining therapeutic efficacy, thus significantly reducing manufacturing time and cost
3Loss of time
If allogeneic donor-derived T cells are used to avoid patient-specific manufacturing, then manufacturing time is reduced, but graft-versus-host disease risk increases due to TCR recognition of host histocompatibility antigens
Solution Approach 1:
The patent extracts and disrupts the TCR complex components (TCR alpha chain, TCR beta chain, and beta-2 microglobulin) from the allogeneic T cells, effectively removing the harmful TCR-mediated recognition capability that causes GVHD. This allows allogeneic T cells to be used without the risk of TCR-mediated rejection while maintaining other therapeutic functions
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 efficient and specific gene editing in primary human T cells, achieving up to 80% disruption of TCR expression and reducing off-target effects, thereby enhancing the safety and efficacy of CAR T cell therapies while minimizing manufacturing time and costs.
Implementation Method 1
CRISPR/Cpf1 technology is facilitating genome engineering in many cell types including T cells
Implementation Method 2
Full depletion of TCR α,β chains and beta-2 microglobin has to be achieved in order to generate truly universal CART cells
Data Source
AI summary
The present invention includes compositions and methods for modifying primary T cells. In one aspect, the invention comprises administering to a cell a stem-loop Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA (st-crRNA) and a Cpf1 enzyme.