Development of an oncolytic mammalian orthoreovirus expressing the near-infrared fluorescent protein iRFP720

Fluorescence-guided surgery (FGS) is a useful method for removing invasive tumor tissues. For this, near-infrared (NIR) fluorescence probes are suitable for visualizing cancer cells due to their low autofluorescence, and an oncolytic mammalian orthoreovirus (MRV) expressing an NIR fluorescent protein is expected to be a novel tool for FGS. In this study, we identified the optimal insertion site of the NIR fluorescent protein gene iRFP720 (915 nt) in the MRV genome. We constructed genome plasmids for the L1, M1, and S2 segments, where a gene cassette comprising iRFP720 and T2A self-cleaving peptide was inserted in the 5′ or 3′ region of each segment. Through virus recovery, the recombinant MRV with the gene cassette at the M1 segment’s 3′ end, T3D-L(M1/3′iRFP720), was capable of replication and passaging with bright NIR fluorescence. However, the replication of T3D-L(M1/3′iRFP720) was approximately 1,000-fold lower than that of the wild-type virus. T3D-L(M1/3′iRFP720) production improved due to the transfection of a fusion-associated small transmembrane protein gene of fusogenic reovirus. Further, fluorescence signals were detected in T3D-L(M1/3′iRFP720)-infected human gastric and pancreatic cancer cells. Thus, the M1 segment’s 3′ end tolerates the expression of the long iRFP720 gene, which may propel the development of recombinant MRV vectors for FGS.