Validation of target genes involved in myeloid immunosuppression for the generation of novel RNA-loaded nanosystems to enhance the efficacy of immunotherapy in Glioblastoma

Glioblastoma, or grade IV glioma, is the most aggressive form of glioma and it has high invasiveness. The standard care of treatment consists of an initial surgical resection, followed by radiation therapy and Temozolomide chemotherapy. However, the prognosis of patients remains poor and new treatments are highly needed. The initial clinical trials with immune checkpoint inhibitors in GBM have been unfortunately disappointing. The reduced efficacy of immunotherapy is likely due to the paucity of T cells and to the immunosuppressive microenvironment of GBM created by tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). Our team is part of a European consortium that aims at finding an innovative therapeutic approach for GBM by the modulation of the tumor microenvironment. In particular, we seek to enhance the efficacy of radiotherapy and anti-PD-1 immunotherapy by the local delivery of RNA-loaded nanomedicines called RAINs (radiotherapy-activated immunizing niches) that will both reduce myeloid-induced immunosuppression and attract and activate cytotoxic T effector cells. The aim of this thesis project is to select and validate suitable target genes, highly expressed by the myeloid compartment of GBM microenvironment, that will be used to generate siRNA-loaded polymeric nanoparticles to be delivered to the tumor resection cavity in preclinical GBM models. In parallel, the first prototype of polymeric nanoparticles will be tested in vitro on a mouse cell line of GBM for the evaluation of its cytotoxicity and transfection efficacy. Finally, in the absence of an approved in vivo model of GBM, a novel in vitro model of myeloid cells differentiated with the tumor-condition medium of GBM cells will be phenotypically characterized and evaluated for the expression of the targets of interest. After the selection of some candidate genes based on the literature and on publicly available single-cell RNAseq data on GBM microenvironment, we selected 10 relevant targets expressed by monocytic/myeloid cells and confirmed their expression in two in vitro models of inhibitory myeloid cells: bone marrow-derived macrophages (BM-MOs) and bone marrow-derived myeloid-derived suppressor cells (BM-MDSCs). From this initial screening, four highly expressed genes (Lgals9, Vsir, Cebpb, Lgals1) were chosen and their expression was confirmed also in two immortalized cell-lines, MSC-1 and MSC-2. These cells will be used to test the effect of the in vitro silencing of the targets on the suppressive function of myeloid cells on T cell proliferation. We were also able to verify the relevance of the chosen targets in a GBM-induced model of primary myeloid cells that likely mimics the in vivo tumor microenvironment. Finally, we had the chance to evaluate the cytotoxicity of the first ZE-NPs that will be optimized by our collaborators in the next months.