T-cell-specific systemic delivery of therapeutic siRNAs for GvHD therapy

• Applicant: Dr. Reka Haraszti
• Subject Area: Pharmacology ; Hematology, Oncology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 565874664

This proposal aims to develop T-cell specific delivery systems for systematic siRNA use, enabling a promising novel therapeutic approach for graft-versus-host-disease (GvHD). Importantly, the proposed therapy is designed to preserve the graft-versus-leukemia (GvL) effect – essential for the cure of stem cell transplanted patients – which is compromised by current approved therapies. The first goal is to create a reporter system that activates upon successful siRNA delivery, allowing assessment of siRNA efficacy and specificity in T cells. This will be achieved using a dCas9-sgRNA and Cre recombinase competition mechanism. The sensor will utilize a fluorescent reporter for in vivo imaging. A reporter cassette with a floxed STOP codon preceding the reporter gene ORF will be designed. Co-expression of Cre and a sgRNA targeting a modified LoxP site will occur, where the sgRNA blocks Cre binding, preventing recombination. siRNAs targeting the sgRNA will degrade it, freeing the site for Cre, which then excises the STOP codon, activating the reporter. Then we will a develop T cell targeting siRNA delivery platform inspired by HIV biology. Specifically, we will engineer virus like particles (VLPs) with HIV-mimetic targeting ligands that bind CD4 and CCR5. We will screen both natural and artificial (i.e. antibody fragments) targeting ligands, and optimize the transmembrane domain in the construct. Covalently conjugated cholesterol will load chemically modified siRNAs onto VLPs. This delivery platform will then be tested in a xenogeneic GvHD mouse model. In the first step, dose finding studies will be conducted in humanized mice expressing the initially developed SWICTH-ON siRNA sensor in T cells. Then our previously developed siRNA cocktail (directed against RAN, WAPAL and AURKA) will be formulated into the novel VLPs and tested for efficacy in the same xenogeneic GvHD mouse model. Once efficacy is confirmed, safety will be assessed in a modified version of the model incorporating GvL effects Potential challenges have been identified, with alternative strategies proposed to ensure the success of each aim. This proposal seeks to develop an innovative solution for targeted siRNAs delivery to T cells, enabling a novel siRNA-based GvHD therapy. This approach could eliminate the need for current unspecific GvHD treatments, which are only effective in 30% of cases and pose significant toxicity risks. The novel siRNA delivery platform could potentially be adapted to other T-cell-mediated diseases, such as autoimmune conditions and immune dysregulation in cancer.

• DFG Programme: Research Grants