Multiplex engineering using microRNA-mediated gene silencing in CAR T cells

Area of Science:

• Immunology
• Cell Therapy
• Molecular Biology

Background:

• Multiplex gene-edited CAR T-cell therapies face challenges, including oncogenic risks from DNA double-strand breaks.
• Targeted microRNAs (miRNAs) present a safer, tunable alternative for gene silencing, avoiding DNA editing.
• This study explores miRNA-based multiplex gene silencing in CAR T cells.

Purpose of the Study:

• To demonstrate multiplex gene silencing using an optimized miRNA approach in CAR T cells.
• To compare the efficacy and safety of miRNA-mediated gene silencing against CRISPR/Cas9 gene editing.
• To evaluate the antitumor activity and immune evasion capabilities of miRNA-silenced CAR T cells.

Main Methods:

• Engineered miRNA-expressing cassettes into M5CAR lentiviral vectors for multiplex gene silencing (TCR, MHC-I).
• Compared miRNA-silenced (S) M5CAR T cells with CRISPR/Cas9 knockout (KO) M5CAR T cells.
• Assessed antitumor activity in vitro and in vivo in pancreatic ductal adenocarcinoma models.

Main Results:

• Silenced M5CAR T cells exhibited comparable or superior antitumor functionality to KO cells.
• In vivo studies showed enhanced tumor control, persistence, and metastasis prevention with S M5CAR T cells.
• In vitro assays revealed increased resistance to alloreactive NK cells and PBMCs.

Conclusions:

• Titratable multiplex gene silencing via miRNAs provides a viable alternative to gene editing for CAR T cells.
• This miRNA strategy offers potential advantages in potency, persistence, metastasis prevention, and immune evasion.
• It may overcome tumor-induced immunosuppression while mitigating risks associated with DNA double-strand breaks.