recombineering-based-manufacturing-engineered-viral-vectors-for

Recombineering-based Manufacturing of Engineered Viral Vectors for Research and Therapy

The increasing demand for engineered viral vectors in both basic and translational research has underscored the need for flexible, rapid, and scalable ...

CEINGE Biotecnologie Avanzate Franco Salvatore S.C.aR.L.

University of Naples Federico II

ImGen-T Srl

Different innate immune pathways converge to Stimulator of interferon genes (STING) and trigger type I interferon responses after recognition of abnormal nucleic acids in the cells. This non-redundant function renders STING a major player in immunosurveillance, and an emerging target for cancer and infectious diseases therapeutics. Beyond somatic mutations that often occur in cancer, the human gene encoding STING protein, TMEM173 (STING1), holds great genetic heterogeneity; R232, HAQ (R71H-G230A-R293Q) and H232 are the most common alleles. Although some of these alleles are likely to be hypomorphic, their function is still debated, due to the available functional assessments, which have been performed in biased biological systems. Here, by using genetic background-matched models, we report on the functional evaluation of R232, HAQ and H232 variants on STING function, and on how these genotypes affect the susceptibility to clinically relevant viruses, thus supporting a potential contributing cause to differences in inter-individual responses to infections. Our findings also demonstrate a novel toll-like receptor-independent role of STING in modulating monocytic cell function and differentiation into macrophages. We further supported the interplay of STING1 variants and human biology by demonstrating how monocytes bearing the H232 allele were impaired in M1/M2 differentiation, interferon response and antigen presentation. Finally, we assessed the response to PD-1 inhibitor in a small cohort of melanoma patients stratified according to STING genotype. Given the contribution of the STING protein in sensing DNA viruses, bacterial pathogens and misplaced cancer DNA, these data may support the development of novel therapeutic options for infectious diseases and cancer.

The common H232 STING allele shows impaired activities in DNA sensing, susceptibility to viral infection, and in monocyte cell function, while the HAQ variant possesses wild-type properties.

Systems biology has been applied at the multi-scale level within the cancer field, improving cancer prevention, diagnosis and enabling precision medicine approaches. While systems biology can expand the knowledge and skills for oncological treatment, it also represents a challenging expedition due to cancer complexity, heterogeneity and diversity not only between different cancer indications, but also in its evolution process through space and time. Here, by characterizing the transcriptional perturbations of the tumor microenvironment induced by oncolytic, we aimed to rationally design a novel armed oncolytic herpes virus. We found that intratumor oncovirotherapy with HSV-1 induces T-cell activation signatures and transcriptionally activates several costimulatory molecules. We identified differentially expressed costimulatory receptors and binding partners, where inducible co-stimulators (ICOS) resulted in the potentially most beneficial targeted therapy. Through an ex-vivo transcriptomic analysis, we explored the potential of arming an oncolytic virus as a combination therapy strategy; in particular, we engineered a targeted herpes virus encoding ICOSL (THV_ICOSL), which resulted in a significant improvement in tumor size control compared to unarmed parental virus. Also, combination with a PD-1 inhibitor enhanced antitumor efficacy as predictable by upregulation of PD-1 and ligands pair (PD-L1/PD-L2) upon oncolytic virus injection. Generation of the human version of this virus encoding hICOSL orthologue effectively and specifically activated human T cells by triggering the ICOS pathway. Our data support the data-driven generation of armed oncolytic viruses as combination immunotherapeutic with checkpoint inhibitors.

Integrating system biology and intratumor gene therapy by trans-complementing the appropriate co-stimulatory molecule as payload in oncolytic herpes virus.

Antisense oligonucleotides (ASOs) are short, synthetic sequences designed to specifically target RNA target molecules to modulate their translation and protein expression. To enhance their stability, chemically modified ASOs have been developed. Over the decades, peptide nucleic acids (PNAs) have emerged as powerful tools in molecular biology, proving their ability in gene knockdown, as well as therapeutic or diagnostic applications. In this study, we sought to explore the potential of PNAs to inhibit RNA translation through a novel mechanism of action. To enhance translation inhibition, we designed a new class of PNAs able to mediate target RNA circularization. These PNAs bind the 5' end of the RNA, interacting with CAP and 5'UTR structures, while simultaneously binding to the poly-A tail at the opposite end of the same RNA molecule. Our findings suggest target RNA circularization and inhibition of protein translation across multiple models. We established a dose-dependent effect of the circularizing PNAs on target RNA translation. Overall, our results reveal that this dual-targeting approach strongly inhibits RNA translation compared to binding solely to the 5'UTR, paving the way to the potential development and therapeutic application of this novel class of ASO PNAs.

Article	Year
The common H232 STING allele shows impaired activities in DNA sensing, susceptibility to viral infection, and in monocyte cell function, while the HAQ variant possesses wild-type properties. Scientific reports\| PubMed ID: 37945588	2023
Integrating system biology and intratumor gene therapy by trans-complementing the appropriate co-stimulatory molecule as payload in oncolytic herpes virus. Cancer gene therapy\| PubMed ID: 38839891	2024
Peptide nucleic acid-mediated circularization of target RNA as tool to inhibit translation. International journal of biological macromolecules\| PubMed ID: 40118420	2025

Emanuele Sasso

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