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

Giulia Golinelli^1,2*John Scholler¹Audrey Roussel-Gervais³Antonija Šakić³Sten Ilmjärv³Decheng Song¹Khatuna Gabunia¹Mei Ji¹Ting J Fan¹Aasha Gupta¹Mansi Deshmukh¹Abdulla Berjis^1,4Riccardo Cuoghi Costantini⁵Kimberly Apodaca¹Neil Sheppard¹Sven Kili³Massimo Dominici²Marco Alessandrini³Carl H June^1,6,7Bruce L. Levine^1*

• ¹Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States
• ²Laboratory of Cellular Therapy, Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy
• ³Antion Biosciences SA, Geneva, Switzerland
• ⁴School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, United States
• ⁵Unit of Clinical Statistics, University Hospital of Modena, Modena, Italy
• ⁶Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, United States
• ⁷Parker Institute for Cancer Immunotherapy at University of Pennsylvania, University of Pennsylvania, Philadelphia, United States

Multiplex gene edited CAR T cell therapies face challenges, including potential oncogenic risks from double-strand breaks. Targeted microRNAs (miRNAs) may offer a safer alternative for functional and tunable gene silencing without DNA editing. As a proof of concept for multiplex gene silencing, we employed an optimized miRNA backbone and gene architecture to silence TCR and MHC-I in mesothelin-directed CAR (M5CAR) T cells, comparing efficacy to CD3ζ and β2M CRISPR/Cas9 knockout (KO) cells. miRNA-expressing cassettes were incorporated into M5CAR lentiviral vectors for combined gene augmentation. Antitumor functionality against in vitro and in vivo models of pancreatic ductal adenocarcinoma was maintained. Silenced (S) M5CAR T cells controlled tumor growth in vivo comparably, if not better than KO cells, with higher persistence and improved metastasis prevention. Enhanced resistance to alloreactive NK cells and PBMCs in vitro was also observed. Titratable multiplex gene silencing offers an alternative to gene editing for CAR T cells to enhance potency, overcome tumor-induced immunosuppression, and for allogeneic products, evade immune rejection.