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298 A miRNA-based shRNA platform for the easy, safe, and effective generation of allogeneic CAR T-cells
  1. Matteo Rossi,
  2. Fanny Huberty,
  3. Jerome Marijsse,
  4. Thuy Nguyen,
  5. Celine Jacques-Hespel and
  6. Eytan Breman
  1. Celyad Oncology, Mont-Saint-Guibert, Belgium
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Chimeric antigen receptor (CAR) T-cells have revolutionized the treatment of hematological malignancies. However, the current autologous approach to CAR T-cell production suffers several drawbacks (difficulty to obtain high quality starting material from patients, limited manufacturing capacity, complex logistics, long vein-to-vein time, high costs), to the point that to date only approximately 10% of eligible patients can receive the treatment. Allogeneic ‘off-the-shelf’ CAR T-cells could help circumvent these issues, but their successful clinical application is hampered by the difficulty to manage graft-versus-host disease (GvHD) and host-versus-graft (HvG). A variety of genetic engineering strategies have been proposed to address these issues, all burdened by the complexity of multiple, potentially risky genome edits. Here we describe a non-gene edited one-step approach for the effective generation of allogeneic CAR T-cells. To this aim, we silenced CD3ζ to target T-cell receptor (TCR) expression, and we downregulated β2M and CIITA to target HLA class I and HLA class II expression, respectively. We aimed at preventing apoptosis induced by CD95L, often expressed by cancer cells, by knocking down CD95.

Methods A microRNA (miRNA)-based short hairpin RNA (shRNA) platform was developed, to allow for the tunable modulation of multiple target genes simultaneously. The platform was equipped with shRNA-derived guide sequences (shGuides) targeting CD3ζ, β2M, CIITA and CD95 simultaneously, and combined with an anti-CD19 CAR. Each shRNA was screened and assessed functionally in vitro.

Results The miRNA-derived shRNA platform targeted against CD3ζ, β2M, CIITA and CD95 in the CAR construct led to a substantial downregulation of TCR, HLA class I, HLA class II and CD95. This, in turn, granted the engineered CAR T-cells protection in in vitro assays mimicking GvHD, HvG and CD95L-induced autophagy, ultimately leading to significantly improved CAR T-cell survival. Notably, thanks to the tunability of gene expression allowed by our platform, we were able to achieve functionally relevant downregulation of HLA class I to avoid recognition by donor T-cells, ensuring at the same time sufficient expression to let CAR T-cells escape NK recognition, without the need of further editing (e.g., HLA E overexpression).

Conclusions These data validate our technology for the effective introduction of multiple functionally relevant edits in CAR T-cells. Moreover, we show in vitro proof-of-concept that our approach can improve allogeneic CAR T-cell viability by allowing evasion from GvHD, HvG and CD95L-induced autophagy. Our technology stands out as an easy, safe, and effective strategy for the generation of allogeneic CAR T-cells.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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