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In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System
Published on: January 9, 2020
Adeno-associated virus-clustered regularly interspaced short palindromic repeats/cas9‑mediated ovarian cancer
Tamaki Yahata1, Saori Toujima2, Izumi Sasaki3
1Department of Obstetrics and Gynecology, Wakayama Medical University, 811-1 Kimiidera, Wakayama, 641-8509, Japan. yahata@wakayama-med.ac.jp.
Abstract:
The response rate of antibody therapy targeting immune checkpoint molecules in ovarian cancer is insufficient. This study aimed to develop a novel gene immunotherapy model targeting programmed death ligand 1 (PD-L1) in vivo in ovarian cancer using adeno-associated virus (AAV)-clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and investigate its efficacy. In vitro, we produced PD-L1-AAV particles to knock out PD-L1. PD-L1-AAV particles were transduced into the murine ovarian cancer cell line ID8. PD-L1 expression at the cellular level was significantly decreased following treatment with PD-L1-AAV particles compared with control-AAV particles. In the peritoneal dissemination model, the survival time was significantly longer in the PD-L1-AAV particles intraperitoneally injected group than that in the control group. Furthermore, intratumoral lymphocyte recruitment was analyzed by immunohistochemistry, and the number of intratumoral CD4+ and CD8+ T cells was significantly higher, whereas that of Foxp3+ Treg cells was significantly lower in the PD-L1-AAV particles injected group than in the control group. No severe adverse events in normal organs, such as the lungs, spleen, liver, and kidney, were observed. These results suggest that PD-L1-targeted therapy by genome editing using AAV-CRISPR/Cas9 is a novel gene-immune therapeutic strategy for ovarian cancer.
Insights
This study introduces a new gene therapy for ovarian cancer using adeno-associated virus (AAV)-CRISPR/Cas9 to target programmed death ligand 1 (PD-L1), improving survival and immune response.
Area of Science:
- Oncology
- Immunotherapy
- Gene Therapy
Background:
- Antibody therapy for ovarian cancer targeting immune checkpoints has limited efficacy.
- Programmed death ligand 1 (PD-L1) is a key immune checkpoint molecule implicated in tumor immune evasion.
Purpose of the Study:
- To develop and evaluate a novel in vivo gene immunotherapy model for ovarian cancer.
- To target programmed death ligand 1 (PD-L1) using adeno-associated virus (AAV)-CRISPR/Cas9 gene editing.
Main Methods:
- Production of PD-L1-AAV particles for gene editing.
- In vitro transduction of murine ovarian cancer cells (ID8) with PD-L1-AAV.
- In vivo assessment in a murine peritoneal dissemination model.
- Immunohistochemical analysis of intratumoral lymphocyte populations.
Main Results:
- Significant reduction in PD-L1 expression in ID8 cells treated with PD-L1-AAV.
- Increased survival time in mice treated with PD-L1-AAV via intraperitoneal injection.
- Enhanced infiltration of CD4+ and CD8+ T cells, with a decrease in Foxp3+ Treg cells within tumors.
- No observed severe adverse events in major organs.
Conclusions:
- AAV-CRISPR/Cas9-mediated PD-L1 targeting represents a promising novel gene-immune therapeutic strategy for ovarian cancer.
- This approach effectively enhances anti-tumor immunity and improves survival in preclinical models.
- The therapy demonstrates a favorable safety profile in normal tissues.
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