{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T18:20:09Z","timestamp":1790274009541,"version":"4.1.0"},"reference-count":34,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2025,2,15]],"date-time":"2025-02-15T00:00:00Z","timestamp":1739577600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Celyad Oncology SA"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>With the success of chimeric antigen receptor (CAR) T-cell therapy in B-cell malignancies, efforts are being made to extend this therapy to other malignancies and broader patient populations. However, limitations associated with the time-consuming and highly personalized manufacturing of autologous CAR T-cells remain. Allogeneic CAR T-cell approaches may overcome these challenges but require further engineering to reduce their alloreactivity. As a means to prevent graft-versus-host disease (GvHD) of allogeneic CAR T-cells, we have selected a micro RNA (miRNA)-based short hairpin RNA (shRNA) targeting CD3\u03b6 which efficiently downregulates the expression of the T-cell receptor (TCR) below detection level. We generated allogeneic anti-B-cell maturation antigen CAR T-cells (CYAD-211) that co-express an anti-CD3\u03b6 miRNA-based shRNA within the CAR construct which efficiently inhibited TCR-mediated signaling in vitro and GvHD in vivo. CYAD-211 was subsequently evaluated in a Phase-I clinical trial (NCT04613557), in patients with relapsed or refractory multiple myeloma. No signs of GvHD were observed despite evidence of engraftment, demonstrating efficient downregulation of the TCR. Our data provide proof of concept that a non-gene-edited technology can generate fully functional allogeneic CAR T-cells, without any signs of GvHD. However, further engineering of the CAR T-cells is needed to improve their persistence and long-term activity.<\/jats:p>","DOI":"10.3390\/ijms26041658","type":"journal-article","created":{"date-parts":[[2025,2,17]],"date-time":"2025-02-17T03:41:47Z","timestamp":1739763707000},"page":"1658","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Clinical Proof-of-Concept of a Non-Gene Editing Technology Using miRNA-Based shRNA to Engineer Allogeneic CAR T-Cells"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9230-3855","authenticated-orcid":false,"given":"Caroline","family":"Lonez","sequence":"first","affiliation":[{"name":"Celyad Oncology, 1435 Mont-Saint-Guibert, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-1422-7408","authenticated-orcid":false,"given":"Jennifer","family":"Bols\u00e9e","sequence":"additional","affiliation":[{"name":"Celyad Oncology, 1435 Mont-Saint-Guibert, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fanny","family":"Huberty","sequence":"additional","affiliation":[{"name":"Celyad Oncology, 1435 Mont-Saint-Guibert, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thuy","family":"Nguyen","sequence":"additional","affiliation":[{"name":"Celyad Oncology, 1435 Mont-Saint-Guibert, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"C\u00e9line","family":"Jacques-Hespel","sequence":"additional","affiliation":[{"name":"Celyad Oncology, 1435 Mont-Saint-Guibert, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1951-6715","authenticated-orcid":false,"given":"Sebastien","family":"Anguille","sequence":"additional","affiliation":[{"name":"Division of Hematology, University Hospital Antwerp (UZ Antwerp), 2650 Antwerp, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anne","family":"Flament","sequence":"additional","affiliation":[{"name":"Celyad Oncology, 1435 Mont-Saint-Guibert, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3562-7781","authenticated-orcid":false,"given":"Eytan","family":"Breman","sequence":"additional","affiliation":[{"name":"Celyad Oncology, 1435 Mont-Saint-Guibert, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,2,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Mitra, A., Barua, A., Huang, L., Ganguly, S., Feng, Q., and He, B. 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