Anti-VSV-N Hybridoma [21H5]

Noninvasive dual-imaging methods that provide an early readout on tumor permissiveness to virus infection and tumor cell death could be valuable in optimizing development of oncolytic virotherapies. Here, we have used the sodium iodide symporter (NIS) and I-125 radiotracer to detect infection and replicative spread of an oncolytic vesicular stomatitis virus (VSV) in VSV-susceptible (MPC-11 tumor) versus VSV-resistant (CT26 tumor) tumors in BALB/c mice. In conjunction, tumor cell death was imaged simultaneously using technetium (Tc-99m)-duramycin that binds phosphatidylethanolamine in apoptotic and necrotic cells. Dual-isotope single-photon emission computed tomography/computed tomography (SPECT/CT) imaging showed areas of virus infection (NIS and I-125), which overlapped well with areas of tumor cell death (Tc-99m-du-ramycin imaging) in susceptible tumors. Multiple infectious foci arose early in MPC-11 tumors, which rapidly expanded throughout the tumor parenchyma over time. There was a dose-dependent increase in numbers of infectious centers and Tc-99m-duramycin-positive areas with viral dose. In contrast, NIS or duramycin signals were minimal in VSV-resistant CT26 tumors. Combinatorial use of NIS and Tc-99m-duramycin SPECT imaging for simultaneous monitoring of oncolytic virotherapy (OV) spread and the presence or absence of treatment-associated cell death could be useful to guide development of combination treatment strategies to enhance therapeutic outcome.

Lentiviral vectors (LVs) pseudotyped with the measles virus hemagglutinin (H) and fusion (F) glycoproteins have been reported to more efficiently transduce hematopoietic stem and progenitor cells (HSPCs) compared with vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped LVs. However, a limit to H/F LV use is the low titer of produced vector. Here we show that measles receptor (CD46) expression on H/F transfected HEK293T vector-producing cells caused adjacent cell membrane fusion, resulting in multinucleate syncytia formation and death prior to peak vector production, leading to contaminating cell membranes that co-purified with LV. H/F LVs produced in CD46 null HEK293T cells, generated by CRISPR/Cas9-mediated knockout of CD46, produced 2-fold higher titer vector compared with LVs produced in CD46(+) HEK293T cells. This resulted in approximately 2- to 3-fold higher transduction of HSPCs while significantly reducing target cell cytotoxicity caused by producer cell contaminates. Improved H/F LV entry into HSPCs and distinct entry mechanisms compared with VSV-G LV were also observed by confocal microscopy. Given that vector production is a major source of cost and variability in clinical trials of gene therapy, we propose that the use of CD46 null packaging cells may help to address these challenges.