Reprogramming intratumoral Tregs by morpholino-mediated splicing of FOXP3 for cancer immunotherapy

Regulatory T cells (Tregs) represent a primary barrier to the development of effective anti-tumor immunity. In the clinic, Tregs are a major contributor to the resistance of immune checkpoint blockade (ICB). Preclinical studies have reported complete tumor regression upon Treg depletion in mice. However, systemic depletion of Tregs is detrimental and sometimes lethal as it causes immune-related adverse effects, such as autoimmune diseases. Here, we report that reprogramming Treg functions, by shifting the expression of FOXP3 from its full-length isoform (FOXP3FL) to a short isoform with exon 2 skipped (FOXP3dE2), promotes the tumor-killing activity of T effectors while limiting the adverse effects of Treg depletion. FOXP3dE2 mRNA expression in triple negative breast cancer tissue positively correlates with overall survival of the patients. Mice expressing only the FOXP3dE2 isoform are resistant to the development of various types of tumors, while the littermate wildtype (WT) mice expressing FOXP3FL isoform exhibit significant tumor growth. Intratumoral injection of FOXP3dE2-directing morpholino (dE2 MO) in WT mice markedly increased IFN-.+ CD4 and CD8 T cells in tumors and consequently inhibited the tumor growth. We developed a novel mouse strain with a humanized Foxp3 exon 2 capable of expressing both FOXP3 isoforms through alternative splicing. This mouse strain showed better antitumor immunity than WT mice and dE2 MO treatment can further enhance their antitumor activity. The antitumor activity of dE2 MO was also confirmed using autologous tumor-infiltrating T cells and patient-derived tumor organoids. Taken together, our results suggest that enhancing FOXP3dE2 isoform expression will reprogram tumor infiltrating Tregs and convert these immune suppressive Tregs to helper-like T cells, thus promoting antitumor immunity. EO771 cancer cells (1 million/mouse) were implanted orthotopically into the fourth mammary gland of 6- to 8-week-old female C57BL6 wildtype mice or Foxp3dE2 mice. Tumor infiltrating lymphocytes (TILs) were isolated at day 10 post tumor inoculations for single-cell sequencing and TCR sequencing.