CD3 Zeta-Deficient CAR Fragment Integration for Allogeneic Therapy
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Solution Overview
Problem
Current CAR-T cell therapy for cancer faces challenges such as complexity in production, high costs, severe side effects, inefficient gene transfer, and risk of graft-versus-host disease due to the use of viral vectors and large gene constructs, particularly in allogenic cell products.
Innovation Solution
A nucleic acid construct integrating a CD3 zeta-deficient chimeric antigen receptor (CAR) fragment into the endogenous CD3 zeta/CD247 gene, allowing for intrinsic regulation and expression, reducing TCR complex formation, and enabling efficient manufacturing of allogeneic CAR-expressing cells without viral vectors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If viral vectors and large gene constructs are used for CAR-T cell therapy, then CAR expression can be achieved, but production complexity and costs increase
Solution Approach 1:
The invention divides the CAR construct into two parts: a coding sequence region that is small and easy to transfer, and a regulatory region that is integrated into the endogenous CD3 zeta gene promoter. This segmentation allows the coding sequence to be efficiently transferred without viral vectors while the regulatory control is provided by the integrated promoter, reducing production complexity.
Solution Approach 2:
The invention merges the CAR coding sequence with the endogenous CD3 zeta gene promoter by integrating the construct at the CD3 zeta locus. This combination allows the CAR expression to be controlled by the native promoter, eliminating the need for separate viral vector-based regulatory elements and simplifying the overall construct.
2Reliability
If viral vectors are used for gene transfer, then CAR expression can be achieved, but side effects like cytokine storms and graft-versus-host disease increase
Solution Approach 1:
The invention extracts the CAR coding sequence from the viral vector-based system and integrates it directly into the endogenous CD3 zeta gene. This extraction eliminates the harmful effects of viral vectors (cytokine storms, graft-versus-host disease) while maintaining CAR expression through the integrated construct at the CD3 zeta locus.
Solution Approach 2:
The endogenous CD3 zeta promoter provides self-regulated expression of the CAR construct. The promoter naturally controls when and where the CAR is expressed, eliminating the need for external viral vector-based regulatory mechanisms that cause harmful side effects. The system serves itself through the native regulatory elements.
3Reliability
If large gene constructs are used for CAR-T cell therapy, then CAR expression can be achieved, but gene transfer efficiency decreases
Solution Approach 1:
The invention segments the gene construct into a small coding sequence region and a regulatory region. The coding sequence is optimized for efficient transfer and integration, while the regulatory control is provided by the integrated promoter. This segmentation dramatically improves gene transfer efficiency compared to using large complete CAR constructs.
4Reliability
If allogeneic CAR-T cells are produced with TCR complex formation, then CAR expression can be achieved, but graft-versus-host disease risk increases
Solution Approach 1:
The invention extracts and eliminates the TCR complex formation by integrating the CAR construct at the CD3 zeta locus, which is a component of the TCR complex. This extraction prevents the formation of functional TCRs on the cell surface, thereby eliminating the risk of graft-versus-host disease in allogeneic CAR-T cell therapy while maintaining CAR expression.
Data Source
AI summary
A nucleic acid construct for targeting and integrating a CD3 zeta-deficient chimeric antigen receptor (CAR) fragment into an endogenous CD3 zeta/CD247 gene of a host genome. Also disclosed is a genetically modified human cell expressing an exogenous nucleic acid sequence encoding a CD3 zeta deficient CAR fragment, integrated in-frame into the endogenous CD3 zeta/CD247 gene for gene fusion, to form a functional CAR including an exogenous CAR fragment fused with an endogenous CD3 zeta domain.