Abstract
Photodynamic and sonodynamic therapy (PDT, SDT) are modern, non-invasive treatments applicable to different types of cancer. PHOTOGLIOBLASTER aims to develop innovative, minimally invasive strategies for the efficient and selective delivery of photo- or sono-activable sensitizers to tumor cells using recombinant viral vectors for the treatment of Glioblastoma (GBM), using two approaches. The first strategy will arm a neuro-attenuated oHSV-1 with the protein KillerRed (KR) under the control of a tumor specific promoter, to clear those oHSV-1 infected cells that have escaped from viral killing with PDT. The second strategy is based on the M13 phage, engineered to target cells expressing the glioma-specific variant of the EGFR receptor and rigged with hundreds of sensitizer molecules, excitable with deep penetrating infrared light or ultrasound. Successful results are expected to pave the road for innovative PDT/SDT cancer therapeutics with broad translational potential.
Results achieved
The first approach focused on the use of oncolytic viruses (OVs), which have emerged as versatile anticancer agents for tumors with poor clinical outcomes. OVs preferentially replicate in malignant cells by exploiting defects in tumor antiviral responses, leading to direct tumor cell lysis and stimulation of antitumor immune responses. Most OVs are derived from attenuated human viruses or nonpathogenic viruses from other species. Oncolytic herpes simplex virus type 1 (oHSV1), for instance, is commonly engineered through deletion of the γ34.5 gene to eliminate neurovirulence. Talimogene laherparepvec (T-VEC), an oHSV1 variant lacking γ34.5 and Us12, was the first OV approved by both the FDA and EMA for the intratumoral treatment of unresectable melanoma (Kaufman HL, et al. Front Mol Biosci. 2022). In addition, T-VEC encodes granulocyte–macrophage colony–stimulating factor (GM-CSF) to enhance antitumor immune activation. Several oHSV1-based vectors are currently being evaluated in clinical trials for GBM. While early clinical studies have demonstrated the safety and biological activity of oHSV1 following intracranial administration, direct delivery remains a significant barrier to widespread clinical use. Invasive procedures such as stereotactic or intrathecal injections limit treatment frequency and scalability. To overcome these limitations, cell-based delivery systems have been proposed. In this strategy, OVs are loaded ex vivo into migratory carrier cells, which can then be systemically administered and selectively home to tumors (Reale A, et al. Pharmaceutics. 2021). In the context of GBM, effective carrier cells must also be capable of crossing the blood–brain barrier (BBB). Monocytes, which account for approximately 10% of circulating leukocytes, represent promising carrier candidates due to their ability to infiltrate the central nervous system and differentiate into tumor-associated macrophages (TAMs). These cells can be isolated from peripheral blood, infected ex vivo with oHSV1, and reinfused as “Trojan horse” carriers. Despite this potential, monocytes have been only minimally explored as OV carriers and have not previously been used with oHSV1. Importantly, although wild-type HSV-1 can infect monocytes, viral replication is inefficient—an advantageous feature, as carrier cells must remain viable and motile until reaching the tumor site. We previously demonstrated that oHSV1-infected human monocytes migrate toward breast cancer cells in vitro and toward head and neck squamous cell carcinoma cells in an in ovo chorioallantoic membrane (CAM) model (Reale A, et al. Int J Mol Sci. 2023). Within the PHOTOGLIOBLASTER project, we further showed using a BBB-on-a-chip model that oHSV1-loaded monocytes can successfully traverse the BBB, target human GBM spheroids, and deliver infectious virus while being shielded from neutralization by circulating immunoglobulins (Micheli S, et al. Mater Today Bio. 2025). Several studies have investigated the combination of virotherapy with PDT. Recently, this dual approach was shown to be effective in murine xenograft GBM models using oHSV1 engineered to express KillerRed (KR), a genetically encoded photosensitizer. Upon illumination with 540–590 nm light, KR generates ROS, enabling spatially controlled photodynamic virotherapy and providing opportunities for combination immunotherapy. Within the framework of PHOTOGLIOBLASTER, we generated a highly neuroattenuated oHSV1 variant expressing KR to assess the therapeutic potential of combining OVT with PDT for GBM treatment. We demonstrated that oHSV1-KR was neuroattenuated both in vitro and in vivo. We then evaluated the cytolytic activity of the recombinant virus in human and murine GBM cell lines as well as patient-derived GBM cells cultured under both 2D and 3D conditions, including spheroids, organoids, and scaffold-based models, with and without monocyte-mediated delivery. Furthermore, we investigated the extent to which KR photoactivation enhanced viral oncolysis and assessed the synergistic effects of combining oHSV1 with phthalocyanine. Monocytes were confirmed to be an efficient delivery vehicle for oHSV1-KR in both murine and human GBM models across 2D and 3D systems. Finally, in a syngeneic C57BL/6J mouse model, treatment with oHSV1—either administered directly or via monocyte carriers—resulted in extensive tumor necrosis accompanied by infiltration of CD3⁺ immune cells. The second approach focused on bacteriophage M13, because of the increasing interest in phages as well-defined, protein-based scaffolds for the construction of nanostructured functional materials. In fact, in addition to their structural versatility, they can serve as innovative, safe and efficient delivery vectors thanks to their high amenability to genetic engineering, enabling the design of a wide range of targeting functionalities. (Turrini E, et al. Cell Mol Life Sci. 2024). Compared to conventional drug delivery systems, phages exhibit strong target avidity and offer numerous functionalisation sites, resulting in high payload capacity and multivalency. Notably, their ability to be conjugated with photoactive sensitizers endows them with phototoxic properties, making them promising candidates for photodynamic therapy (PDT) applications (Gunaydin G, et al. Front Chem. 2021). Filamentous phages, like M13, can be imagined as long, thin, biological 'nanocarriers' that are about 1000 nm wide and 6 nm thick. Their structure consists of approximately 2,700 copies of the major coat protein (pVIII), which form the filament capsid. Four minor coat proteins (pIII, pVI, pVII and pIX), with five copies each, are located at the phage tips. In this study, we adopted an orthogonal approach to utilise recombinant phages as robust and adaptable platforms for epidermal growth factor receptor (EGFR)-targeted photodynamic therapy (PDT) strategies. (Ulfo L, et al. Nanoscale. 2022). During the project, we successfully engineered and validated a bacteriophage-based platform for targeted photo- and sonodynamic therapy (PSDT) against EGFRvIII-expressing murine glioblastoma. First, we generated and characterized two EGFRvIII-targeted M13 phages. Among the vectors tested, the M13 7D12 nanobody-displaying phage, directed against the wild-type EGFR receptor, demonstrated superior structural stability and targeting efficiency compared to the M13 EGFRvIII construct, leading to its selection for subsequent studies. We then established a syngeneic murine mGBM-vIII glioblastoma model and confirmed stable EGFRvIII expression in vitro. We demonstrated that the selected phage specifically binds EGFRvIII-positive cells, is internalized, and effectively penetrates even 3D tumor spheroids. Rose Bengal (RB) conjugation enabled efficient ROS generation upon laser or ultrasound activation, resulting in significant in vitro cytotoxicity under both PDT and SDT conditions, while maintaining biosafety in the absence of activation. We also demonstrated the in vivo safety of intratumoral and intravenous administration of the targeted phage vector. The phage selectively accumulated within the tumor tissue and persisted over time. Importantly, SDT treatment in combination with M137D12 –RB bioconjugates showed promising results, with a slight increase in median survival, that warrants further investigation in future studies. In conclusion, our results established M137D12 –RB bioconjugates as a promising and safe triggerable oncolytic vector for targeted photo-sono-oncolytic ablation of EGFRvIII-positive glioblastoma, providing a solid foundation for further preclinical optimization and translational development. Final Remarks Overall, the data collected by PHOTOGLIOBLASTER paved the route to novel therapeutic strategies to tackle the burden of glioblastoma. In vitro efficacy of both approaches implemented in the project proved very promising, supporting their translation to more preclinically relevant in vivo models. This experimentation is still ongoing and will be conducted further, in search for much needed clinical solutions to treat patients affected by GBM.Dettagli del progetto
Responsabile scientifico: Alberto Danielli
Strutture Unibo coinvolte:
Dipartimento di Farmacia e Biotecnologie
Coordinatore:
Università degli Studi di PADOVA(Italy)
Contributo totale Unibo: Euro (EUR) 54.223,00
Durata del progetto in mesi: 24
Data di inizio
12/10/2023
Data di fine:
28/02/2026
