Allogeneic cell therapy stands out as a potent strategy for leukemia treatment. However, allogeneic T cells can trigger the desired Graft-versus-Leukemia (GvL) effect and the unwanted Graft-versus-Host (GvH) Disease. Conventional  GvHD drugs are non-selective, simultaneously inhibiting both GvH and GvL effects, resulting in widespread toxicities and complications in patients. This study explores the potential of sequence-specific siRNAs to temporarily modify the phenotype of allogeneic T cells, thereby enhancing the efficacy of cell-based therapies.

We designed siRNAs targeting four genes (AURKA, WAPAL, KIF15, RAN) that were shown to be upregulated during GvHD. Through screening, we identified best performing siRNAs and subjected them to functional GvH and GvL assays to assess their ability to inhibit allogeneic T cell proliferation. Treatment with siRNA targeting RAN demonstrated dose-dependent inhibition of T cell proliferation in response to both allogeneic (activated by major-mismatched dendritic cells, up to 80%, p=0.01, N=5) and non-specific (activated by CD2/CD3/CD28 beads, 60%, p<0.0001, N=3) stimuli. Singular siRNAs against WAPAL, AURKA, and KIF15 showed no effect on T cell proliferation. Surprisingly, combinations of three siRNAs targeting different genes resulted in potent and specific T cell inhibition, achieving up to 75% inhibition of T cell proliferation upon allogeneic stimulus (p= 0.007, N= 3), while not impacting T cell proliferation upon non-specific stimulus. Treatment with siRNAs did not compromise therapeutically essential GvL effect of T cells. Furthermore, our siRNAs induced a shift in T cell phenotype towards regulatory T cells.

Our data provide proof-of-principle for siRNA mixes as a precision immunomodulatory therapy for GvHD.