(F-215) Seven-Fold Increase Observed in AAV Transduction of Hepatocytes In Vitro due to Electric Pulse Delivery

Introduction:: The liver's vital role in metabolism and blood processing has driven interest in liver-directed gene therapies for genetic diseases such as hemophilia, phenylketonuria, and familial hypercholesterolemia. The use of adeno-associated virus (AAV) vectors for gene delivery is a promising approach due to their ability to transduce dividing and non-dividing cells with long-lasting, non-mutagenic effects. However, challenges in scaling AAV use to therapeutic dose levels include the triggering of an immune response and high costs. We hypothesize that applying electric field pulses (EP) to hepatocytes can increase AAV transduction efficiency, reducing dosage, costs, and side effects while improving safety. While EP has been explored for non-viral plasmid vector enhancement, its impact on AAV vector transduction remains relatively unexplored. In this in vitro study, we investigate the effect of EP on the transduction efficiency of liver-tropic AAV serotype 8 (AAV8) in the Hep G2 human hepatoma cell line. Our findings demonstrate that EP significantly increases AAV8 transduction in hepatocyte-derived cells, suggesting potential for EP-enhanced gene therapy as a novel approach to reducing the AAV dosage required to achieve clinical efficacy.


Materials and Methods::
To investigate the effect of EP exposure on AAV transduction efficiency, we designed experiments with two treatment groups: NoEP-AAV (control) and EP-AAV. Both groups shared the same multiplicity of infection (MOI) of 2.5x10⁵ (vg/cell), but we only exposed the EP-AAV group to EP. Prior to EP application, we removed the culture medium and rinsed the cells with EMEM (without FBS), and then added AAV suspended in EMEM to individual wells. We delivered EP using a BTX Model ECM 830 Square Wave Electroporation System with parallel-plate adherent cell electrodes separated by a 5-mm gap, and applied as a single 80 ms pulse at 375 V/cm field strength. We incubated the cells at 37°C and 5% CO2 for 12 hours post-treatment, after which we replaced the AAV-containing medium with culture medium (EMEM+FBS). At 48 hours post-treatment, we quantified AAV transduction efficiency by comparing fluorescence intensity between the EP-AAV and NoEP-AAV groups.




Results, Conclusions, and Discussions::
Results




Fluorescence microscopy images reveal that EP exposure indeed enhanced AAV transduction efficiency in hepatocytes. Fig. 1a shows that approximately 15% of cells in the NoEP-AAV group were successfully transduced, as quantified by post-treatment fluorescence. In contrast, Fig. 1b shows that nearly 100% of cells in the EP-AAV group were transduced, with oversaturated fluorescence observed. Additionally, our observations of the complementary phase contrast images consistently showed only viable cells with negligible evidence of cell damage from EP exposure.

Discussions and Conclusions

Our findings show that EP exposure dramatically increases (by more than a factor of seven) AAV transduction efficiency in the Hep G2 cell line. Using a definition of transduction efficiency that combines cell transduction (yes/no) with the extent of transduction per cell (number of gene copies which is correlated with the fluorescence emission intensity), we consider the seven-fold enhancement factor to be an underestimate, given the fact that the cell fluorescence images for the EP exposed cells were consistently over-saturated. This suggests a potential strategy to significantly reduce AAV vector doses, minimizing patient risk, reducing treatment cost, and enhancing efficacy for long-lasting, viral-mediated transduction in clinical gene therapy.






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