PCSK9-Silenced CAR-T Cells for Solid Tumor Immune Escape
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Solution Overview
Problem
Current CAR-T cell therapies face challenges in effectively targeting solid tumors due to the immunosuppressive tumor microenvironment and the inhibitory effects of PCSK9, and there is a need to enhance T-cell function and reduce the demand for peripheral blood mononuclear cells (PBMC) to mitigate graft-versus-host disease and lymphopenia.
Innovation Solution
A method involving the use of miRNA fragments and CRISPR-Cas9 technology to interfere with or knockout the PCSK9 gene in CAR-T cells, enhancing their anti-tumor activity by integrating miRNA into the CAR expression vector and delivering Cas9/sgRNA RNP for rapid and specific gene editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If PCSK9 expression is increased in tumor-infiltrating T-cells, then tumor immune escape is enhanced, but anti-tumor activity is inhibited
Solution Approach 1:
The patent converts the harmful effect of PCSK9 (inhibiting anti-tumor activity) into a beneficial target for intervention. By designing miRNA molecules that specifically target and silence PCSK9 expression in T-cells, the invention transforms the problematic pathway into a therapeutic opportunity, enhancing anti-tumor activity while maintaining immune system functionality.
Solution Approach 2:
The patent changes the expression level parameter of PCSK9 in T-cells from high (inhibitory) to low (enhanced anti-tumor activity). Through miRNA-mediated gene silencing, the invention precisely controls and modulates PCSK9 expression levels, converting the inhibitory state into a therapeutic state that improves CAR-T cell function and immune checkpoint response.
2Adaptability or versatility
If allogeneic CAR-T is used to treat tumor patients, then treatment coverage is expanded, but graft-versus-host disease occurs
Solution Approach 1:
The patent extracts and removes the harmful PCSK9 component from the system. By specifically silencing PCSK9 expression in allogeneic CAR-T cells through miRNA, the invention separates the beneficial anti-tumor activity from the harmful effects that lead to graft-versus-host disease, enabling safer use of allogeneic cell therapies.
3Ease of manufacture
If conventional CAR-T preparation process is used, then standard treatment is provided, but demand for patients' PBMC is high
Solution Approach 1:
The patent changes the expression parameter of PCSK9 in CAR-T cells to reduce the functional demand on cell quantity. By enhancing the activity and efficiency of each individual T-cell through PCSK9 silencing, the invention reduces the total number of PBMCs required from patients, making the treatment process less invasive and more feasible.
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 method effectively silences PCSK9 expression, improving CAR-T cell function and tumor treatment efficacy while reducing the required number of PBMCs, thereby enhancing immune checkpoint therapy and minimizing off-target effects.
Implementation Method 1
the technology of artificial miRNA silencing gene is also more and more used in tumor research, this method is based on the mammalian endogenous miRNA precursor as the basic skeleton, and replaces the miRNA sequence in the stem-loop structure with the artificial miRNA sequence partially complementary to the target gene mRNA, so that the generated artificial miRNA acts on the target gene, thereby inhibiting the expression of the target gene
Implementation Method 2
Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) gene editing technology is to identify the target genome sequence through sgRNA and guide Cas9 nuclease to effectively cut the genome to form DNA double-strand breaks, and then realize gene knockout, knock-in, mutation, etc. through the cell's repair mechanism to achieve gene editing purposes
Implementation Method 3
The method of directly transferring ribonucleoprotein (RNP) formed by Cas9 and sgRNA into cells is safer and faster, with lower off-target effect and higher editing efficiency
Data Source
AI summary
A method for preparing enhanced CAR-T-cells based on interfering or knocking out the human PCSK9 gene and an application thereof are provided. The method includes performing the RNA interference (RNAi) for the human PCSK9 gene by the artificial microRNA (miRNA) to prepare the enhanced CAR-T or preparing the enhanced CAR-T by CRISPR-Cas9 gene-editing technology by using sgRNA to knock out the human PCSK9 gene. The method uses PCSK9 gene knockdown element or CRISPR/Cas9 technology to specifically knock down or knock out the expression of PCSK9 in CAR-T-cells, which effectively enhances the therapeutic effect of CAR-T-cells on malignant solid tumors, and can enhance the tumor therapeutic effect of immune checkpoint blockade therapy.