Electric pulse exposure reduces AAV8 dosage required to transduce HepG2 cells

Abstract

We demonstrate that applying electric field pulses to hepatocytes, in vitro, in the presence of enhanced green fluorescent protein (EGFP)-expressing adeno-associated virus (AAV8) vectors reduces the viral dosage required for a given transduction level by more than 50-fold, compared to hepatocytes exposed to AAV8-EGFP vectors without electric field pulse exposure. We conducted 48 experimental observations across 8 exposure conditions in standard well plates. The electric pulse exposures involved single 80-ms pulses with 375 V/cm field intensity. Our study suggests that electric pulse exposure results in enhanced EGFP expression in cells, indicative of increased transduction efficiency. The enhanced transduction observed in our study, if translated successfully to an in vivo setting, would be a promising indication of potential reduction in the required dose of AAV vectors. Understanding the effects of electric field pulses on AAV transduction in vitro is an important preliminary step.

Fig 1. Experimental treatment groups involving hepatocytes exposed to various doses of AAV, AAV* (AAV pre-treated with EP), or plasmids, with or without electric field pulse (EP) exposure (illustrations not drawn to scale).

Fig 2. Fluorescence microscope images show that applying EP in vitro significantly enhanced the AAV transduction of Hep G2 cells.

a Fluorescence microscope image of the NoEP-NoAAV group (control), showing no detectable transduction. b Fluorescence microscope image of the NoEP-HighAAV group showing transduction and expression by ~15% of cells; c Fluorescence microscope image of the EP-HighAAV group showing transduction and expression by ~100% of cells. We applied the same microscope camera exposure settings to all groups.

Fig 3. Fluorescence microscope images of the groups NoEP-NoAAV, NoEP-HighAAV, and EP-LowAAV.

a b c Fluorescence microscope images of the groups NoEP-NoAAV (control), NoEP-HighAAV, and EP-LowAAV, respectively. d Fluorescence intensity histograms for the groups NoEP-NoAAV (grey), NoEP-HighAAV (blue), and EP-LowAAV (red). The horizontal axis represents the rescaled fluorescence intensity and the vertical axis shows the percentage of pixels at each specific fluorescence intensity. We collected data from three fields of view per image, for two distinct experiments, yielding a total of six data sets. The mean values of the six data sets at each intensity are plotted as solid lines and the range between the minimum and maximum values is indicated by the shaded area between the dashed lines.

Fig 4. Histogram of group NoEP-HighAAV, NoEP-HighAAV*, and NoEP-HighPlasmid.

The horizontal axis represents the rescaled fluorescence intensity, and the vertical axis shows the percentage of pixels at the specific rescaled fluorescence intensity. We collected data from 3 fields of view per image, for two separate repetition experiments, yielding a total of 6 data sets. The mean values of the six data sets at each intensity are plotted.