Engineered AAV2.7m8 Serotype Shows Significantly Higher Transduction Efficiency of ARPE-19 and HEK293 Cell Lines Compared to AAV5, AAV8 and AAV9 Serotypes.

The level of transduction efficiency of the target retinal cells affects the choice of AAV serotype and the outcome of gene replacement therapy for inherited retinal diseases. This study focused on the tropism and transduction efficiency of AAV2.7m8-, AAV5-, AAV8-, and AAV9-GFP in ARPE-19 and HEK293 cells. Fluorescence intensity was assessed bi-hourly by means of IncuCyte S3 live imaging microscopy. Within 12 h, AAV2.7m8 demonstrated the highest transduction efficiency at four viral concentrations of 1-, 3-, 6-, and 8 × 104 VG/cell in a dose-dependent manner, followed by AAV5 in ARPE-19 and AAV9 in HEK293 cells. The transduction efficiency of AAV2.7m8 at a dose of 6 × 104 VG/cell was 21, 202, and 323 times higher in ARPE-19 cells and 324, 100, and 52 times higher in HEK293 cells compared to AAV5, AAV8, and AAV9, respectively. This trend remained for 4 days at all viral concentrations, as additionally shown by flow cytometry. At a dose of 6 × 104 VG/cell, AAV2.7m8 (97% GFP-positive cells, GFP +) was nearly two and 10 times as efficient as AAV5 (52% GFP+) and AAV9 or AAV8 (both 9%), respectively, in ARPE-19 cells. In HEK293 cells, 95% of AAV2.7m8-, 26% of AAV9-, 17% of AAV8-, and 12% of AAV5-transduced cells were GFP-positive.

Development of Recombinant Oncolytic rVSV-mIL12-mGMCSF for Cancer Immunotherapy.

Anti-cancer therapy based on oncolytic viruses (OVs) is a targeted approach that takes advantage of OVs' ability to selectively infect and replicate in tumor cells, activate the host immune response, and destroy malignant cells over healthy ones. Vesicular stomatitis virus (VSV) is known for its wide range of advantages: a lack of pre-existing immunity, a genome that is easily amenable to manipulation, and rapid growth to high titers in a broad range of cell lines, to name a few. VSV-induced tumor immunity can be enhanced by the delivery of immunostimulatory cytokines. The targeted cytokine delivery to tumors avoids the significant toxicity associated with systemic delivery while also boosting the immune response. To demonstrate this enhanced effect on both tumor growth and survival, a novel recombinant VSV (rVSV)-mIL12-mGMCSF, co-expressing mouse IL-12 (interleukin-12) and GM-CSF (granulocyte-macrophage colony-stimulating factor), was tested alongside rVSV-dM51-GFP (rVSV-GFP) that was injected intratumorally in a syngeneic in vivo C57BL/6 mouse model infused subcutaneously with B16-F10 melanoma cells. The pilot study tested the effect of two viral injections 4 days apart and demonstrated that treatment with the two rVSVs resulted in partial inhibition of tumor growth (TGII of around 40%) and an increased survival rate in animals from the treatment groups. The effect of the two VSVs on immune cell populations will be investigated in future in vivo studies with an optimized experimental design with multiple higher viral doses, as a lack of this information presents a limitation of this study.