{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T17:51:31Z","timestamp":1788285091708,"version":"build-2803163510"},"reference-count":46,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,8,21]],"date-time":"2025-08-21T00:00:00Z","timestamp":1755734400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>Multiplex gene-edited chimeric antigen receptor (CAR) T-cell therapies face significant challenges, including potential oncogenic risks associated with double-strand DNA breaks. Targeted microRNAs (miRNAs) may provide a safer, functional, and tunable alternative for gene silencing without the need for DNA editing.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      As a proof of concept for multiplex gene silencing, we employed an optimized miRNA backbone and gene architecture to silence T-cell receptor (TCR) and major histocompatibility complex class I (MHC-I) in mesothelin-directed CAR (M5CAR) T cells. The efficacy of this approach was compared to CD3\u03b6 and \u03b22-microglobulin (\u03b22M) CRISPR\/Cas9 knockout (KO) cells. miRNA-expressing cassettes were incorporated into M5CAR lentiviral vectors, enabling combined gene silencing and CAR expression. Antitumor activity was evaluated using\n                      <jats:italic>in vitro<\/jats:italic>\n                      assays and\n                      <jats:italic>in vivo<\/jats:italic>\n                      pancreatic ductal adenocarcinoma models.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Silenced (S) M5CAR T cells retained antitumor functionality comparable to, and in some cases exceeding, that of KO cells.\n                      <jats:italic>In vivo<\/jats:italic>\n                      , S M5CAR T cells achieved tumor control with higher persistence and superior metastasis prevention.\n                      <jats:italic>In vitro<\/jats:italic>\n                      assays demonstrated enhanced resistance to alloreactive natural killer (NK) cells and peripheral blood mononuclear cells (PBMCs).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>Titratable multiplex gene silencing via targeted miRNAs offers an alternative to gene editing for CAR T cells, with potential advantages in potency, persistence, metastasis prevention, and immune evasion for allogeneic products. This strategy may overcome tumor-induced immunosuppression while avoiding the risks associated with DNA double-strand breaks.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fimmu.2025.1647433","type":"journal-article","created":{"date-parts":[[2025,8,21]],"date-time":"2025-08-21T04:12:12Z","timestamp":1755749532000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":8,"title":["Multiplex engineering using microRNA-mediated gene silencing in CAR T 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