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Installation of Dominant-Negative Mutations in FAS and TGFβR2 via Base Editing in Primary T Cells

Bryce J Wick1,2,3, Mitchell G Kluesner4,5, Nicholas J Slipek1,2,3

  • 1Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.

Biorxiv : the Preprint Server for Biology
|September 26, 2025
PubMed

Insights

Base editing installs dominant-negative mutations in T cells, enhancing their function against solid tumors by overcoming immunosuppression from FASL and TGFβ signaling. This approach improves CAR-T cell efficacy and persistence in challenging tumor microenvironments.

Area of Science:

  • Immunology
  • Molecular Biology
  • Cancer Therapy

Background:

  • Adoptive cell transfer (ACT) shows promise for B-cell malignancies but struggles with solid tumors due to the immunosuppressive tumor microenvironment (TME).
  • FAS ligand (FASL) and transforming growth factor beta (TGFβ) are key mediators of T cell dysfunction within the TME.
  • Current strategies involve overexpressing dominant-negative (dn) receptors, but endogenous gene targeting offers advantages for multiplex editing.

Purpose of the Study:

  • To utilize base editing (BE) for installing dominant-negative mutations in endogenous FAS and TGFβ receptor 2 (TGFβR2) genes within primary human T cells.
  • To assess the impact of these BE-engineered mutations on T cell resistance to immunosuppressive signals and their anti-tumor efficacy in solid tumor models.
  • To compare the efficacy of BE-mediated gene editing with traditional lentiviral cDNA overexpression methods.

Main Methods:

  • Primary human T cells were engineered using base editing to introduce naturally occurring dominant-negative mutations into the FAS and TGFβR2 genes.
  • In vitro assays were performed to evaluate T cell survival, proliferation, and resistance to FAS and TGFβ signaling.
  • Chimeric antigen receptor (CAR)-T cells engineered with BE-installed dn TGFβR2 or dn FAS were assessed for cytotoxicity, persistence, and exhaustion markers.
  • Functional assays compared BE-engineered CAR-T cells against lentiviral-engineered counterparts.

Main Results:

  • BE-engineered T cells demonstrated resistance to the pro-apoptotic and anti-proliferative effects of FAS and TGFβ.
  • CAR-T cells with BE-installed dn TGFβR2 or dn FAS exhibited enhanced cytotoxicity.
  • dn TGFβR2 CAR-T cells showed improved persistence and reduced expression of exhaustion markers compared to controls.
  • BE-engineered dn CAR-T cells outperformed lentiviral-engineered cDNA overexpression systems in several functional assays.

Conclusions:

  • Base editing provides an efficient method for engineering T cells with dominant-negative mutations to counteract immunosuppression in the TME.
  • This strategy enhances the anti-tumor efficacy, persistence, and reduces exhaustion of CAR-T cells against solid tumors.
  • BE-mediated endogenous gene editing represents a promising platform for developing next-generation T cell therapies for solid tumors.
Keywords:
CAR TDominant-negative mutationsT cellsbase editorscancer immunotherapy

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