EXOSOMES AS A STROMAL-TARGETING EXTENSION OF ONCOLYTIC VIRUS THERAPEUTICS

Abstract

Background & Aim: Background

Oncolytic viruses (OVs) selectively replicate in and kill cancer cells. OVs can be used as a gene delivery vector for selective expression of therapeutics in the tumour microenvironment (TME). One current barrier of OV-mediated therapy in solid tumours is the poor viral spread due to the extracellular matrix produced from stromal cells such as cancer-associated fibroblasts (CAFs).

Exosomes are lipid-coated nanoparticles which can be engineered to transport therapeutics between donor and recipient cells.

Aim

To enhance the spread of therapeutics in situ, we are developing an exosome-targeting system where DNA coding for a tripartite fusion protein (consisting of a targeting protein (anti-FAP scFv), a scaffold protein, and a therapeutic protein (cytosine deaminase)) is delivered by an oncolytic adenovirus. The fusion protein is synthesized and loaded into exosomes for delivery to surrounding potentially non-infected tumour and stromal cells such as CAFs.

Methods, Results & Conclusion: Methods

Exosomes were isolated via size exclusion chromatography from adenocarcinoma cells stably transduced with the tripartite fusion protein and characterized according to MISEV guidelines. Real-time exosome uptake into cancer cells and CAFs was monitored using a split nanoluciferase reporter system. Nano-flow cytometry and a FlipGFP-reporter system were used to evaluate the functionality and targeting potential of the anti-FAP scFv exosmes. Cell viability assays were used to quantify the ability of exosomes to deliver cytosine deaminase to cancer cells and CAFs.

Results

Exosomes containing a subunit of split nanoluciferase were capable of being taken up in real-time and delivering functional cargo into the cytoplasm of recipient tumour cells expressing the other subunit. A functional anti-FAP scFv was capable of being expressed on exosomes, and exosomes armed with cytosine deaminase resulted in a significant decrease in tumour cell viability following the addition of 5-fluorocytosine.

Conclusion

Exosomes can be engineered to selectively package therapeutics and deliver cargo to recipient cells resulting in target cell death. The cytotoxicity of the oncolytic virus expressing the tripartite fusion protein is currently being evaluated using these methods in vitro and ex vivo. Concentrating the production of bioengineered EVs directly in the TME provides a novel approach to overcome the biodistribution and rapid EV clearance challenges that limit the clinical potential of therapeutic Exosomes/EVs.