Vitiello, Adriana (2018) Generation and characterization of HSV-1 based oncolytic viruses towards an innovative therapeutic approach for TNBC. [Ph.D. thesis]
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Abstract (italian or english)
Immunotherapy, mainly based on checkpoint inhibitors or Chimeric Antigen Receptor (CAR) T cells, is providing a major breakthrough in the pharmacological therapy of cancer. However, many solid tumors surrounded by an immunosuppressive tumor microenvironment (TME), like triple negative breast carcinoma (TNBC), do not have a cytotoxic T lymphocyte infiltrate that can be activated by checkpoint inhibitors (“cold” tumors). Oncolytic viruses (OVs) are able to selectively replicate in cancerous cells, which are often defective in antiviral response pathways. OVs provide a powerful proinflammatory stimulus in the TME, thus subverting its immunosuppressive features. Therefore, OVs are very promising agents for the immunotherapeutic treatment of immunologically “cold” tumors. At the same time, OVs can be “armed” to express therapeutic molecules that enhance their efficacy. Herpes simplex virus type 1 (HSV-1) proved to be a suitable platform for the generation of OVs, due to its large dsDNA genome, which allows extensive manipulation and insertion of therapeutic genes, to its lytic activity and good safety profile. HSV-1 derived OVs usually carry deletions of the γ34.5 gene, which is essential for neurovirulence. The HSV-1 based talimogene laherparepvec (T-VEC) is the only OV approved for clinical use (metastatic melanoma) in the EU and the USA so far.
METHODS Bacterial artificial chromosome (BAC) mutagenesis in an appropriate strain of Escherichia coli, that is characterized by the inducible expression of a recombinase system, was adopted for the manipulation of the HSV-1 genome. In particular, mutagenesis was performed on a previously available Δγ34.5 HSV-1 BAC. Viral stocks were then reconstituted by lipofectamine transfection of purified BAC DNA into 293T cells. We tested the selectivity and the replicative capacity of the recombinant viruses in a panel of human mammary cancer cell lines as well as in both tumoral and non-tumoral murine mammary organoids. The efficiency of expression of transgenes was also evaluated by different methods.
RESULTS AND CONCLUSIONS The Us12 gene, encoding the ICP47 protein, was succesfully deleted from the parental BAC. The Us12 deletion enhances MHC-I antigen presentation by infected cells, and shifts the expression kinetics of the Us11 gene from late to immediate early, boosting viral replication without affecting safety. First of all a recombinant virus was generated by the insertion within the UL55-UL56 intergenic region of the enhanced green fluorescent protein (EGFP) gene. The recombinant virus showed specificity for tumoral organoids and efficiently killed cancer cells. Based on these findings, two additional recombinant viruses were developed by replacing the EGFP gene with a sequence encoding either i) a single chain antibody (scAb) targeting C-C Chemokine Receptor Type 4 (CCR4) or ii) the solubile Programmed cell Death-1 (sPD1) protein. Both recombinant viruses efficiently replicated in the tested cell lines. Exogenous genes were also expressed. To address the immunological complexity of the TME, we are inserting further therapeutic genes, including human IL-12, and FMS-like tyrosine kinase 3 ligand (Flt3L) in the Δγ34.5/ΔUs12 BAC. Next steps will be a deep in vitro characterization of the developed OVs and testing viral therapeutic efficacy on an appropriate mouse model.
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