HPK1 Gene Knockout in CAR-T Cells for Solid Tumor Therapy
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Solution Overview
Problem
Current cancer immunotherapy methods, such as CAR-T cell therapy, face challenges in effectively targeting solid tumors due to T cell depletion, decreased activity, and insufficient tumor killing ability, particularly in overcoming T cell exhaustion and depletion.
Innovation Solution
Genetic disruption of the HPK1 gene in immune cells, such as T cells, using agents like CRISPR-Cas9 to induce knockout or reduction of HPK1 expression, combined with the introduction of recombinant receptors like chimeric antigen receptors (CARs), to enhance cytotoxicity and persistence while reducing exhaustion markers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CAR-T cell therapy is used to treat solid tumors, then T cell activity is enhanced, but T cell depletion and exhaustion occur leading to reduced efficacy
Solution Approach 1:
The patent extracts and removes the harmful HPK1 protein from T cells through CRISPR/Cas9-mediated gene knockout. By specifically targeting and eliminating HPK1 expression in CAR-T cells, the invention resolves the contradiction by removing the molecular mechanism that causes T cell exhaustion while preserving the therapeutic anti-tumor function of the CAR-T cells
Solution Approach 2:
The patent changes the molecular parameter of HPK1 expression levels in T cells by completely knocking out the HPK1 gene. This parameter change transforms T cells from an exhausted state (high HPK1 expression) to an activated persistent state (zero HPK1 expression), thereby extending T cell duration of action while maintaining tumor killing ability
2Power
If T cell activity is enhanced for better tumor killing, then cytotoxicity increases, but T cell exhaustion markers increase leading to depletion
Solution Approach 1:
The patent converts the harmful effect of HPK1 (causing exhaustion) into a benefit by completely eliminating HPK1 expression. The CRISPR/Cas9 system creates double-strand breaks in the HPK1 gene, and the resulting NHEJ repair introduces frameshift mutations that permanently disable HPK1. This transformation eliminates the source of exhaustion markers while preserving enhanced cytotoxicity
Solution Approach 2:
The patent performs preliminary genetic modification of T cells before CAR transduction by knocking out the HPK1 gene. This preliminary action ensures that T cells are pre-programmed to resist exhaustion before they are activated with CARs, preventing the development of exhaustion markers while maintaining high cytotoxicity throughout the therapeutic process
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
The approach significantly enhances the cytotoxicity and persistence of immune cells, improves their ability to infiltrate tumors, and reduces exhaustion markers like PD-1, TIM-3, and Lag-3, leading to improved antitumor efficacy comparable to or exceeding PD-1 gene knockout strategies.
Implementation Method 1
gRNA having a targeting domain that is complementary with a target domain of the HPK1 gene
Implementation Method 2
creation of a double stranded break (DSB) in the HPK1 gene (e.g., in the first or second exon), which is repaired by non-homologous end joining (NHEJ) that effects insertions and deletions (indels) in the HPK1 gene
Data Source
AI summary
Provided is a gRNA targeting HPK1 and a method for editing HPK1 gene. The method can knock out the T cell HPK1 gene, enhance the T cell killing activity, increase the Th1 cytokine level of peripheral blood mononuclear cells, and knock out of the T cell HPK1 gene can also down-regulate the expression of PD-1 and TIM3 on the T cell surface and can inhibit the T cell depletion.