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. 2024 Apr 30;19(4):e0298866. doi: 10.1371/journal.pone.0298866

Electric pulse exposure reduces AAV8 dosage required to transduce HepG2 cells

Yizhou Yao 1,*, Robert W Holdcraft 2, Susan C Hagness 1, John H Booske 1
Editor: Chen Ling3
PMCID: PMC11060518  PMID: 38687720

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.

Introduction

The liver performs many vital metabolic and blood-processing functions. This centrality to health has motivated investigations of liver-directed gene therapies, which modify the DNA content of hepatocytes to cure numerous life-threatening genetic diseases such as hemophilia A and B, phenylketonuria, acute intermittent porphyria, familial hypercholesterolemia, and primary hyperoxaluria [1–6]. Effective liver-directed gene therapies feature three attributes: efficient targeting of the hepatocytes, a stable vector genome, and persistent high-level expression [1]. The use of adeno-associated viruses (AAV) as gene transfer vectors is a promising strategy for viral-mediated in vivo gene therapy and has demonstrated long-lasting therapeutic effects for a number of metabolic diseases [7–11]. AAV vectors are advantageous for transducing both dividing and non-dividing cells while sustaining long-term, episomal (nonmutagenic) transgene expression [12]. Multiple AAV serotypes have been identified to date, which allows an extensive variety of diseases to be targeted [13, 14] due to AAV’s diverse cell tropism. Non-human primate studies have shown that AAV serotype 8 (AAV8) can transduce cells in liver, muscle, and cardiac tissues while maintaining transgene expression for over five years post-delivery [15, 16].

While AAV has been used successfully to transduce cells in small animals, its scale-up to larger animals [17] and humans has been hindered to date by several challenges. First, a high dose of viral vectors can trigger potentially severe immune responses [18]. Second, adaptive immunity may limit subsequent treatment efficacy [19]. Finally, the required AAV dose in clinical trials is cost-prohibitive [20]. Therefore, lowering the required AAV dose could significantly improve safety and reduce the cost of AAV-vector-mediated, liver-directed gene therapies.

We hypothesized that applying electric field pulses to hepatocytes at pulse durations and intensity levels that avoid irreversible electroporation or thermal effects could increase AAV transduction efficiency. An increased transduction efficiency would, in turn, reduce the therapeutic dosage and cost, minimize side effects, and improve the safety of AAV-mediated gene therapy treatments. Although electric pulsing (EP) has been used to improve the transfection and/or transduction efficiency of non-viral plasmid vectors [21–23], the mechanism and potential effects of EP directly on AAV have not been extensively investigated. To the best of our knowledge, there are no prior published investigations of the direct effects of EP on viral vector transduction efficiency. In this in vitro study, we investigated the impact of EP on transduction efficiency in the Hep G2 human hepatoma cell line by AAV8 (with known affinity for liver tissue). While other serotypes might exhibit better inherent transduction rates in vitro, the principle we aimed to investigate was whether EP could offer a significant enhancement in transduction efficiency, regardless of the baseline performance of the chosen serotype. Our results indicate the capacity of EP to augment viral vector transduction through the specific example of AAV8 in a hepatocyte-derived cell line. These results demonstrate an in vitro enhancement of viral vector transduction efficiency, incentivizing subsequent investigations of whether similar enhancements exist in vivo.

Materials and methods

Cell culture and AAV vector

We used the human hepatoma cell line Hep G2 (American Type Culture Collection, Manassas, VA, USA) to study the transduction efficiency of AAV8. Hepatoma cell lines are commonly used in vitro as alternatives to primary human hepatocytes [24]. We cultured Hep G2 cells at 37°C and 5% CO2 in Eagle’s Minimum Essential Medium (EMEM, American Type Culture Collection, Manassa, VA, USA) supplemented with 10% fetal bovine serum (FBS), hereafter referred to as the culture medium. Approximately 24 hours before conducting experiments, we seeded 1x 106 viable Hep G2 cells into tissue-culture-treated 12-well plates (Corning, NY, USA) in 1000 uL culture medium and grown to at least 80% confluency per well. Viable cell counts were determined by manual cell counts using Trypan Blue (Lonza, Walkersville, MD, USA) exclusion. The AAV8 vector used in these experiments was scAAV8.CMV.EGFP.WPREs (Packgene Biotech, Worcester, MA, USA) which contains enhanced green fluorescent protein (EGFP) as the reporter and provides a convenient way to measure transduction efficiency via fluorescence microscopy. This vector is simply referred to as “AAV” in most instances throughout the remainder of this manuscript.

Overview of experimental treatment groups

We designed an initial round of experiments with five treatment combinations of EP and AAV dosages to test the hypothesis that EP exposure improves AAV transduction efficiency. Cells were either exposed to EP, denoted as the EP group, or not exposed to EP, labeled NoEP. Furthermore, cells were exposed to a high, low, or no AAV dose, labeled HighAAV, LowAAV, or NoAAV, respectively. The five treatment combinations included two without EP (NoEP-NoAAV and NoEP-HighAAV) and three involving EP (EP-NoAAV, EP-LowAAV, and EP-HighAAV). The effect of EP on the transduction efficiency of AAV was quantified by comparing fluorescence intensity in the EP versus NoEP treatment groups.

Based on observations from the initial experiments described above, a second round of experiments was conducted with three additional treatment groups. In one treatment, cells were exposed to a high AAV* dose, where AAV* refers to vector that was exposed to an electric pulse in a cuvette prior to being introduced to cells. This treatment scenario is denoted as NoEP-HighAAV*. In two other groups, labeled NoEP-HighPlasmid and EP-LowPlasmid, cells were exposed to either high or low doses of naked plasmid DNA which contained the same EGFP expression cassette present in the AAV vector. In another treatment scenario, labeled NoEP-Medium*-HighAAV, cells were exposed to a high AAV dose in a culture medium that was pre-treated with EP, but neither the cells nor the AAV were exposed to EP. These additional combinations allowed us to isolate whether EP enhanced AAV transduction efficiency through effects on the cells, the medium, or the AAV virions. We provide more detailed descriptions of treatment combinations in the following subsections. Each experimental treatment was performed in duplicate. Within each well, we analyzed three non-overlapping fields of view in the fluorescence images, resulting in a total of six data sets collected per group. The chosen fields of view were systematically and consistently selected across all samples to provide a representative view of the region between the electrodes. Our preliminary experiments and validations have indicated that these three fields of view show consistent results with broader sampling and thus are indeed representative.

Experimental conditions to test the EP-enhanced transduction hypothesis

Fig 1 illustrates eight of the nine experimental treatment groups. The AAV dosing for the initial five treatment groups used to test the EP-enhanced transduction hypothesis was as follows:

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 1

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  • NoEP-NoAAV: No treatment control

  • NoEP-HighAAV: MOI (multiplicity of infection) = 2.5×105 (vg/cell).

  • EP-NoAAV: EP-only control

  • EP-LowAAV: MOI = 0.25×105 (vg/cell).

  • EP-HighAAV: MOI = 2.5×105 (vg/cell).

Immediately before the EP application, the culture medium was removed from the cells and all wells were rinsed with EMEM (without FBS). AAV suspended in EMEM or AAV-free EMEM was then added to individual wells. Electrical pulses were delivered using a BTX Model ECM 830 Square Wave Electroporation System with parallel-plate adherent cell electrodes (Harvard Apparatus, Holliston, MA, USA) separated by a 5-mm gap. The parallel-plate electrodes were oriented perpendicular to the cell growth surface on the bottom of the well. The bottoms of both electrodes included small, insulated feet that suspended the lower ends of the electrodes 0.3 mm above the cell monolayer. We delivered a single 80 ms pulse with 375 V/cm field strength [25] between the electrodes in all of the EP groups. Only the cells positioned at the center of the wells and located in the 5 mm gap between the electrodes received the full pulse exposure. Cells located outside of the active zone between the electrodes in each well received a lower or negligible electric field exposure and were not labelled as EP-exposed. All cells were then incubated at 37°C and 5% CO2 for 12 hours post-treatment. The EMEM was subsequently replaced with culture medium (EMEM+FBS).

Conditions for the additional experiments to isolate the effect of EP

The NoEP-HighAAV* experiments involved pre-exposing AAV virions to a single, 80-ms, 375 V/cm pulse using a cuvette. After removing the cell culture medium and rinsing the adherent cell layer with EMEM, we added a dose of the resulting AAV* (pre-exposed to EP) with an MOI of 2.5×105 vg/cell to the cell layer. For the NoEP-HighPlasmid experiments, we exposed the cells to plasmids having the same EGFP expression cassette as AAV (Plasmid Maxi-prep of scAAV.CMV.EGFP.WPREs, Packgene Biotech, Worcester, MA, USA) with an MOI of 2.5×105 plasmid copies/cell. For the EP-LowPlasmid experiments, we exposed cells to plasmids with an MOI of 0.25×105 plasmids/cell and then exposed the cells and plasmids to EP. In the NoEP-HighAAV* or NoEP-HighPlasmid experiments, we incubated both cell groups at 37°C and 5% CO2 for 12 hours to mimic the conditions of the other treatment groups.

Finally, in experiments labeled NoEP-Medium*-HighAAV, we subjected a sample of EMEM in a cuvette to EP using a single 80-ms, 375 V/cm pulse. We then suspended the AAV in the EP-exposed EMEM. After removing the culture medium from the cells and rinsing with EMEM, the suspension of AAV in EP-exposed EMEM was added to the wells at an MOI of 2.5×105 vg/cell. We incubated the group at 37°C and 5% CO2 post-experiment for 12 hours, then replaced the solution with regular culture medium.

Gene expression analysis

Cultured cells were analyzed 48 hours post treatment for EGFP-expression by fluorescence microscopy. This timing allowed for induction of marker gene expression and fluorescence development as cell confluency reached 100%. As illustrated in four of the images of Fig 1, this experimental design precluded delivering the full electric field strength of the EP treatment to all of the cells in the bottom of the wells. Instead, the full (designed) electric field strength was only deliverable to the subset of the cells located centrally between the two electrodes. Cells at the edges of the electrodes would have received a weaker EP exposure from the fringing electric fields. Furthermore, cells “behind” the electrodes would not have received any significant EP exposure. Because of this unavoidable nonuniform spatial distribution of the EP electric field and the subsequent significant cell population outside of the EP zone, cell flow cytometry was not a practical option for quantifying fluorescence expression to characterize the effect of EP on transduction efficiency. It would have been impractical to attempt to only collect cells from the central “EP zone” from each individual well. Therefore, we chose to use fluorescence microscopy.

In addition to visual comparison of gross fluorescence differences between microscopy images, we quantitatively analyzed the fluorescence expression intensity distributions (histograms) as a potential source of further differentiation between cells exposed to AAV and EP from those exposed to AAV alone. Image J, a Java-based image processing program developed at the National Institution of Health (NIH) and the Laboratory for Optical and Computational Instrumentation (University of Wisconsin-Madison), was used to distinguish between fluorescing and non-fluorescing cells. A cell was determined to be fluorescing if its internal fluorescence intensity exceeded the threshold defined by excluding the background pixels. The percentage of fluorescing cells provided a convenient proxy for the estimated transduction efficiency of AAV. We also developed a Matlab script for generating fluorescence histogram plots. The fluorescence histograms that we report here are graphical representations of the number of pixels in the image versus their intensity values. The dispersion of fluorescence intensities provides valuable information about the GFP expression per cell. A higher intensity value corresponds to higher expression inside the cell, as a proxy for higher transduction efficiency. Conventionally, the pixels are grouped by their intensity values assigned by the camera software and the quantity of each reported value is displayed. We rescaled the raw intensity range of the image pixels to range from 0 to 100 to streamline interpretation. Similarly, we replaced the number of pixels at each fluorescence intensity value by a percentage of the pixels (the number of pixels at the specific intensity divided by the overall pixel count). The maximum intensity value of 100 (after rescaling) represents the saturated value where brightness exceeded the resolvable range of the camera’s sensors. We note that the actual intensity value of any pixel recorded as 100 could—and in many cases probably does—exceed the maximum reportable intensity value.

Results

Image-based assessment of AAV cell transduction enhancement via EP

Fig 2 shows representative fluorescence microscopy images, revealing the enhanced transduction efficiency of hepatocytes transduced by AAV via EP exposure. No fluorescence above background was observed in the NoEP-NoAAV control group (Fig 2A). Only ~15% of observed cells in the NoEP-HighAAV group were successfully transduced by AAV as measured by post-treatment fluorescence (Fig 2B). In contrast, nearly 100% of cells in the EP-HighAAV group were transduced, and much of the fluorescence is oversaturated (Fig 2C). The transduction values were obtained using the ImageJ software, as described in the previous gene expression analysis section. These results demonstrated that EP enhanced the AAV transduction of hepatocytes by more than seven-fold compared to AAV transduction without EP.

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

Fig 2

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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.

Analysis of the fluorescence intensity distributions

Oversaturation of the fluorescence microscope image—i.e., fluorescence intensity exceeding the maximum recording limit of the camera by a majority of the cells—in the EP-HighAAV (high MOI) group shown in Fig 2C significantly reduced the useful quantitative information available on marker gene expression. This was an unavoidable consequence of the necessity to maintain consistent microscope settings across all groups for a fair comparison. Therefore, we lowered the AAV dose by ten-fold in the EP-LowAAV group to accommodate the dramatic transduction efficiency enhancement from EP exposure. In Fig 3, we present representative fluorescence microscope images for experiments with this EP-LowAAV group along with the NoEP-NoAAV and NoEP-HighAAV groups. No fluorescence was detectable above background in the NoEP-NoAAV control (Fig 3A). Comparing Fig 3B (NoEP-HighAAV) and 3C (EP-LowAAV) demonstrates that AAV transduction efficiency was dramatically improved by EP exposure, similar to the improvement observed in Fig 2. However, the EP exposure enhancement effect in Fig 3 is still observed in the EP-LowAAV group, despite a 10-fold reduction of the AAV MOI relative to the NoEP-HighAAV group.

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

Fig 3

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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.

The fluorescence intensity histogram in Fig 3D uses the same concept as flow cytometry fluorescence intensity histograms to communicate information about the GFP expression level through the fluorescence intensity distribution. A higher fluorescence intensity value indicates increased GFP expression. The fluorescence from the NoEP-HighAAV group has a narrow histogram span that peaks at a lower intensity level of ~15. The fluorescence from the EP-LowAAV group shows a broader histogram span and has two peaks: one at ~45 and the other at saturated values around 100.

Comparison of AAV* and plasmid results for isolating mechanisms

Fig 4 presents fluorescence microscopy image intensity histograms for the groups NoEP-HighAAV, NoEP-HighAAV*, and NoEP-HighPlasmid. The histograms of the NoEP-HighAAV and NoEP-HighAAV* groups share a similar shape and narrow span and both peak at the low intensity of ~15. Meanwhile, the histograms for the NoEP-HighPlasmid and EP-LowPlasmid experiments indicated no evidence of transduction, as they were identical to the NoEP-NoAAV control group, both in the narrow span and a peak centered at ~10. The NoEP-HighPlasmid and EP-LowPlasmid experiments resulted in no detectable fluorescence above background in the images (not shown), identical to the NoEP-NoAAV image (shown).

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

Fig 4

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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.

Finally, in the cells associated with the NoEP-Medium*-HighAAV experiments, we observed no evidence of enhanced transduction due to pre-exposure of the medium to EP, with results identical to the NoEP-HighAAV and the NoEP-HighAAV* groups.

Measuring transduction efficiency with weighted average cell fluorescence intensity values

We converted the results of Fig 3D into a more conventional comparison of transduction efficiency in terms of a single number per image. Specifically, we calculated the weighted average fluorescence intensity <I> over the distribution. The results of those calculations are provided in Table 1. Higher <I> indicates a higher transduction efficiency. Based on this metric, the EP-LowAAV experiments yielded a greater than four-fold increase in the transduction efficiency over the NoEP-HighAAV experiments. The statement “greater than” is based on the significant number of cells (pixels) registering saturated intensity values of 100 in Fig 3D, which indicate that the true (unsaturated) average should be greater than 59.1, and thus the true (unsaturated) increase factor should be higher than four.

Table 1. Weighted average cell fluorescence distribution intensity for the EP-LowAAV experimental group is at least four-fold higher than that of the NoEP-HighAAV group.

The average intensity <I> is computed from the fluorescence intensity distributions shown in Fig 3D. Note that <I> for the EP-LowAAV group is an underestimation due to the significant number of cells (pixels) registering saturated intensity values of 100 in Fig 3D.

Group <I> ± S.D.
Control 10.1 ± 1
NoEP-HighAAV 16.0 ± 4
EP-LowAAV 59.1 ± 20
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Discussion

Our results demonstrate that exposing hepatocytes to an electric field pulse significantly enhances their transduction efficiency by AAV and could potentially reduce the therapeutic dose of AAV required in liver-directed gene therapies. With identical high AAV doses, the introduction of EP dramatically increased AAV transduction of the Hep G2 cell line, increasing efficiency seven-fold and saturating the fluorescence microscope image relative to the NoEP group (cf. Fig 2). By reducing the AAV dose for the EP-exposed group by a factor of 10 compared to the NoEP group, we significantly reduced the fluorescence image oversaturation to obtain a more quantitative characterization of the effect of EP on transduction efficiency. First, the images in Fig 3B and 3C qualitatively confirm that even with a 10-fold reduced MOI of AAV exposure, EP still provides a dramatic (greater than) four-fold enhancement of AAV transduction over the NoEP case. While further dilution experiments in vitro may provide more precise data regarding the magnitude of the effect EP has on transduction efficiency, our proof-of-concept experiments already confirmed our hypothesis of a strong enhancement effect and satisfied the goals of this study.

Fig 3D reveals further value of analyzing the fluorescence intensity distributions of the microscopy images. It shows that the shape, peak, and span of fluorescence intensity distribution histograms are dramatically different between the NoEP-HighAAV and the EP-LowAAV groups. The broader span and the significantly higher intensity peak of the EP-LowAAV group establishes that the gene expression in group EP-LowAAV is remarkably stronger than NoEP-HighAAV, both in the percentage of transduced cells and the EGFP expression levels. Note that the histogram for the EP-LowAAV group has two peaks, one at ~40 and another at 100. The latter indicates the presence of cells that have even higher expression, beyond the saturated detection limit of 100. Specifically, enhanced GFP expression (in addition to enhanced number of cells transduced) suggests optimistic prospects for long-term, persistent transduction. If one interprets stronger GFP expression as an indicator of more gene products and more episomal genetic insertion, then such a result would enable continued high expression even after multiple cell divisions.

Analysis of the data presented in Table 1 reveals a significant enhancement in transduction efficiency attributable to EP. A detailed examination of the weighted average fluorescence intensities at varying AAV dosages—0.25×105, 1.75×105, and 2.5×105 vg/cell—established a linear relationship with an R-squared value of 0.927, as shown in S1 Fig, in Supporting Information. Applying this linear relationship to the results in Table 1, we conclude that for the non-EP treated cells to reach the same average fluorescence intensity of 59.1 as the EP treated cells, the required AAV dosage would be at least ~13.5×105 vg/cell (compared to 0.25×105 vg/cell for the EP treated cells). Thus, EP treatment has reduced the AAV8 dosage required for the same transduction efficiency by more than 50-fold. These results have important implications for gene therapy research, as they suggest that EP and optimized conditions can lead to improved gene delivery with significantly reduced AAV dosages.

We conducted several experiments to isolate the mechanism behind the phenomenon of EP resulting in higher hepatocyte transduction efficiency by AAV: an effect on the medium, the virions, or the cell membranes. From the NoEP-Medium*-HighAAV experiment results we concluded that EP-enhancement of hepatocyte transduction by AAV was not due to modification of the cell medium by EP exposure. Pre-exposing the medium to EP (without exposing the cells or AAV to EP) yielded transduction outcomes that were identical to conventional experiments that exposed the cells only to AAV vectors without any EP. Ref. [26] reported enhanced gene transfer from pre-exposing cell culture medium to EP. In that case, the authors revealed the enhancement was due to impurity ions generated by an electrochemical reaction between the electrolytic medium and their aluminum electrodes. In contrast, our EP-exposed medium experiments ruled out the possibility that EP altered the medium to enhance transduction. This confirmed our expectation that our use of inert stainless steel electrodes for EP would prevent such an effect from being observed in our experiments. In another set of experiments (referred to as the AAV* group) we applied EP only to the AAV and not to the cells. By applying electric pulses to the AAV virions independently, we aimed to confirm that the virions themselves are not intrinsically altered by the electroporation process to become more effective at transduction. In other words, if there was any enhanced transduction seen after independently electroporating the virions, it could suggest some direct modification or activation of the virions due to electroporation. However, our observations confirm the prevailing understanding: the primary mechanism of increased transduction efficiency with EP is due to its effects on the cell membrane, rather than any inherent change in the virions. The results in Fig 4 show that the fluorescence intensity histograms for the NoEP-HighAAV* cells and the NoEP-HighAAV are essentially identical. This demonstrates that EP did not impact the shape of AAV virions or modify their capsids. If EP did significantly modify the AAV virions, one would expect that the histograms for NoEP-HighAAV and NoEP-HighAAV* would be different in shape or position. The AAV* groups suggest that there are no inherent changes in the AAV virions themselves as a result of EP. Instead, the increased efficiency of AAV8 transduction in HepG2 cells is primarily due to the modifications in the cell membrane induced by EP, which may enhance the cell’s receptivity to AAV entry. In a third group of experiments, we exposed cells to naked plasmid DNA, both with and without EP. The rationale for using a plasmid as a control for AAV genome stems from a test hypothesis that during EP application, the AAV might undergo stress that causes it to release its genome in a form resembling a naked plasmid DNA. By having a plasmid control, we aimed to explore and understand the behavior of unpackaged DNA (akin to potentially decapsulated AAV gene fragments) during EP application. This would illuminate how AAV genomes, in general, might behave under similar conditions. The amounts of plasmid used in both EP treated and untreated groups were kept consistent in terms of gene copy numbers. This was to ensure a fair and accurate comparison between the two groups. Any evidence of transduction in these plasmid experiments fell below the detectable fluorescence thresholds of our camera settings. This ruled out the possibility that our observations of EP-enhanced transduction with AAV vectors were due to the EP forcefully causing ejection of the genome from the capsid and transfecting the cells with vector DNA. This outcome contrasts with observations reported elsewhere of EP-enhanced transfection with naked plasmid DNA (e.g., [27, 28] and references cited therein). However, our results are not inconsistent with [27, 28] because our experimental EP parameters were significantly less energetic than those used in prior plasmid transfection experiments. For example, we used a lower electric field strength (375 V/cm) rather than the more intense electric fields of 400–800 V/cm [27, 28], and we used only a single EP pulse rather than a train of 10 pulses [27, 28]. Nevertheless, our EP parameters were sufficient to induce a remarkable transduction efficiency enhancement with AAV vectors.

In conclusion, we have demonstrated that EP can markedly enhance in vitro AAV transduction of a hepatocyte-derived cell line, effectively reducing the required AAV dosage by more than 50 times to achieve the same level of transduction efficiency. Experiments using AAV virions pre-exposed to EP, plasmid DNA, and medium that was pre-exposed to EP established that the transduction efficiency enhancement was not due to modifications of the AAV virions or the cell culture medium. Rather, this effect is directly dependent upon interaction between the cells and AAV vector in the presence of the electric field, via an as-yet undetermined mechanism. Hypotheses include mechanisms of enhanced endocytosis, such as those elucidated in [28] in their naked DNA experiments, or other electrophoretic effects increasing interaction between the virions and cell membranes. These results suggest a new means to significantly reduce the required dose of AAV vectors, reducing risks to the patient and improving efficacy for successful, long-lasting, virus-mediated transduction in gene therapy.

Supporting information

S1 Checklist. Human participants research checklist.

(DOCX)

pone.0298866.s001.docx (52.7KB, docx)
S1 Fig. Verification of the liner relationship between AAV dosage and fluorescence intensity.

To substantiate the conclusions drawn in our main text regarding the impact of EP on transduction efficiency, we conducted a set of controlled experiments. The objective was to quantitatively establish the relationship between AAV dosage and the resultant transduction efficiency within HepG2. The experiments involved the application of three distinct dosages of AAV: 0.25×105, 1.75×105, and 2.5×105 vg/cell. Post-transduction, the cells were imaged and analyzed to obtain fluorescence intensity measurements, which served as a proxy for transduction efficiency. These intensity values were then normalized and rescaled to create a standard range from 0 to 100, facilitating comparison across varying AAV dosages. A linear regression analysis was performed on the weighted average fluorescence intensities against the corresponding AAV dosages. This analysis yielded an R-squared value of 0.927, indicating a linear relationship between AAV dosage and transduction efficiency.

(TIF)

pone.0298866.s002.tif (113.4KB, tif)

Acknowledgments

Microscopy was performed at the University of Wisconsin-Madison Biochemistry Optical Core, which was established with support from the University of Wisconsin-Madison Department of Biochemistry Endowment.

The authors gratefully acknowledge helpful suggestions from Dr. T. Alam.

Data Availability

All the raw data needed to replicate the results of the study are included as either Supporting Information or uploaded to the Open Science Framework. The URL link to the data in the Open Science Framework is https://osf.io/7xy8n/.

Funding Statement

The author(s) received no specific funding for this work.

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Decision Letter 0

Chen Ling

28 Nov 2023

PONE-D-23-36580Enhancement of AAV vector transduction of hepatocytes via electric pulse exposurePLOS ONE

Dear Dr. Yao,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This research describes an important strategy for improving AAV8 transduction of HepG2 cells by applying a low electric pulse. Authors demonstrate that the increased transduction efficiency is not due to changes to cell media or uptake of AAV vector DNA genome. They also provide compelling evidence for profound (40-fold) increase in AAV transduction. This information could serve as a preliminary step in improving AAV gene delivery to liver with direct translational impact.

There is also in vivo evidence of this enhancement in other organs, for example, “Trans-ocular electric current in vivo enhances AAV[8]-mediated retinal transduction in large animal eye after intravitreal vector administration,” (Transl Vis Sci Technol. 2020 Jun; 9(7): 28). Please note that I have no affiliation with the mentioned laboratory/group.

There are still many questions to be answered: Can this treatment improve transduction efficiency of other AAV serotypes? Can this treatment change AAV serotype tropism? Is this treatment effective in other cells? This paper provides the basis for exploring the effects of low electric pulse in other transduction scenarios.

The manuscript is well-written and well-organized. There is great value in sharing this information with the scientific community. I highly recommend the publication of this manuscript with one minor change: I recommend replacing AAV with AAV8 in your title since other serotypes are not discussed in this manuscript.

Reviewer #2: The author used electrical pulse exposure to stimulate cells and AAV8 carrying the EGFP reporter gene. After two batches of experiments, it was ultimately found that AAV8 can effectively transduce HepG2 cells exposed to electrical pulses compared to the control group.

Major revisions:

1.Compared to AAV8, AAV2 also has good liver targeting. Can the author repeat the same experiment with AAV2 serotype. At the same time, although the fusion degree of cells reached 100% at 48 hours, but for the control group( No EP high/low AAV), the fluorescence at 72 hours would be further enhanced. Therefore, the fluorescence data at 72 hours can also be compared to further clarify the experimental improvement of fluorescence magnification.

2.The author's experimental data has shown that EP can significantly enhance the transduction efficiency of AAV8 on liver cancer cell line HepG2. Can further experiments be conducted using AAV6 serotypes on suspension cell lines such as K562 and THP-1 to further consolidate the conclusion that EP can improve AAV transduction.

3.According to the author, the group that only stimulates cells is labeled as the EP group, while the group that only stimulates AAV is labeled as AAV *. The author's data shows that the group with EP-AAV has the best effect, indicating that the stimulation of cells by EP greatly improves the transduction efficiency of AAV8-EGFP on HepG2. It should be due to some changes in cells that make AAV easier to enter cells, but the author's discussion suggests that the interaction between AAVs leads to this phenomenon. Can the author further explain and present data for the EP AAV * group.

4.For the discussion section, the author speculates that this electrical stimulation may increase the functional interaction between AAVs, but further explanation is needed for what changes occur to cells due to this EP stimulation and where AAVs enter the cells after EP stimulation.

5.The fluorescent images of the transfected plasmid after EP stimulation should also be displayed.

6.A gradient of electricity intensify can be set instead of simply dividing into EP and NoEP.

7.How did you calculate the weighted average fluorescence intensity? Please interpret your formula.

8.The mechanism of the enhanced transduction in molecular level need further research, and you can have a brief discussion on it.

9.Line 309, “enhanced transduction by over 40-fold” doesn’t make sense since the transduction efficiency may not be in proportion to the concentration of AAV8, so you can’t simply multiply these two numbers. Please give more evidence to support this idea.

Minor Concerns:

1.Peaks of the saturated value around 100 needs further research.

2.Line 349, please discuss the affects of impulses on the transduction more in detail.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Negin P. Martin

Reviewer #2: Yes: Chen Ling

**********

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PLoS One. 2024 Apr 30;19(4):e0298866. doi: 10.1371/journal.pone.0298866.r002

Author response to Decision Letter 0

12 Jan 2024

Reviewer #1: This research describes an important strategy for improving AAV8 transduction of HepG2 cells by applying a low electric pulse. Authors demonstrate that the increased transduction efficiency is not due to changes to cell media or uptake of AAV vector DNA genome. They also provide compelling evidence for profound (40-fold) increase in AAV transduction. This information could serve as a preliminary step in improving AAV gene delivery to liver with direct translational impact.

There is also in vivo evidence of this enhancement in other organs, for example, “Trans-ocular electric current in vivo enhances AAV[8]-mediated retinal transduction in large animal eye after intravitreal vector administration,” (Transl Vis Sci Technol. 2020 Jun; 9(7): 28). Please note that I have no affiliation with the mentioned laboratory/group.

There are still many questions to be answered: Can this treatment improve transduction efficiency of other AAV serotypes? Can this treatment change AAV serotype tropism? Is this treatment effective in other cells? This paper provides the basis for exploring the effects of low electric pulse in other transduction scenarios.

The manuscript is well-written and well-organized. There is great value in sharing this information with the scientific community. I highly recommend the publication of this manuscript with one minor change: I recommend replacing AAV with AAV8 in your title since other serotypes are not discussed in this manuscript.

Response:

We thank the reviewer for pointing out the opportunity to clarify the scope of our paper in the title. We have replaced AAV with AAV8 and hepatocytes with HepG2 cells, and modified the title accordingly as follows: Electric Pulse Exposure Reduces AAV8 Dosage Required to Transduce HepG2 Cells.

Reviewer #2: The author used electrical pulse exposure to stimulate cells and AAV8 carrying the EGFP reporter gene. After two batches of experiments, it was ultimately found that AAV8 can effectively transduce HepG2 cells exposed to electrical pulses compared to the control group.


Major revisions:

1.Compared to AAV8, AAV2 also has good liver targeting. Can the author repeat the same experiment with AAV2 serotype. At the same time, although the fusion degree of cells reached 100% at 48 hours, but for the control group( No EP high/low AAV), the fluorescence at 72 hours would be further enhanced. Therefore, the fluorescence data at 72 hours can also be compared to further clarify the experimental improvement of fluorescence magnification.


2.The author's experimental data has shown that EP can significantly enhance the transduction efficiency of AAV8 on liver cancer cell line HepG2. Can further experiments be conducted using AAV6 serotypes on suspension cell lines such as K562 and THP-1 to further consolidate the conclusion that EP can improve AAV transduction.

Response to comment 1 and 2:

The scope of the current study is a proof of concept regarding the efficacy of EP in enhancing AAV transduction, using AAV8 and HepG2 to illustrate the enhancement effect. As suggested by reviewer 1, we have clarified the scope of our study by revising the title of the manuscript.

We agree that it will be valuable to explore additional serotypes and cell lines in future studies, now that we have established the proof of concept. 


3.According to the author, the group that only stimulates cells is labeled as the EP group, while the group that only stimulates AAV is labeled as AAV *. The author's data shows that the group with EP-AAV has the best effect, indicating that the stimulation of cells by EP greatly improves the transduction efficiency of AAV8-EGFP on HepG2. It should be due to some changes in cells that make AAV easier to enter cells, but the author's discussion suggests that the interaction between AAVs leads to this phenomenon. Can the author further explain and present data for the EP AAV * group.

Response:

This is a helpful question because it points to a potential misunderstanding of our original description. We modified the fourth paragraph of the discussion section (starting with line 332 in the revised manuscript) to clarify the prevailing understanding and the purpose of conducting the experiments with AAV8*.  Namely, the primary mechanism behind the increased transduction efficiency observed in the EP-AAV group is attributed to the effects of EP on the cell membrane. The AAV* groups suggest that there are no inherent changes in the AAV virions themselves as a result of EP. Instead, the increased efficiency of AAV8 transduction in HepG2 cells is primarily due to the modifications in the cell membrane induced by EP, which enhance the cell's receptivity to AAV entry. 


4.For the discussion section, the author speculates that this electrical stimulation may increase the functional interaction between AAVs, but further explanation is needed for what changes occur to cells due to this EP stimulation and where AAVs enter the cells after EP stimulation.

Response:

In our discussion, we noted that our observation confirms the prevailing understanding: “the primary mechanism of increased transduction efficiency with EP is due to its effects on the cell membrane”.

Our working hypothesis is that the EP induces nanopores and the resulting membrane stress might create microscopic folds. These folds could potentially act as sites for enhanced endocytosis of nanoparticles like AAV virions. This could explain the observed increase in transduction efficiency when EP, AAV, and the presence of cells are combined.

However, it is premature to add these speculations to this proof of concept paper. Further research is necessary to conclusively determine the molecular changes induced by EP and how these changes facilitate AAV entry into cells.


5.The fluorescent images of the transfected plasmid after EP stimulation should also be displayed.

Response:

To address the reviewer’s request, we are providing the requested image here (please see the attached document Respond to Reviewers)

However, we have decided not to include this image in the revised manuscript. The primary reason for this decision is to avoid redundancy. The histograms for the NoEP-HighPlasmid and EP-LowPlasmid experiments indicated no evidence of transduction. We believe that including it in the paper would consume extra space but not contribute additional insights beyond what has already been described and discussed in the text.


6.A gradient of electricity intensify can be set instead of simply dividing into EP and NoEP.

Response:

We thank the reviewer for suggesting using a gradient of electric pulse intensities. Our current study, however, uses representative EP parameters in the experiments to establish a baseline understanding of EP's impact on AAV transduction – consistent with the goal of this investigation. While we recognize the potential insights from exploring varying EP intensities, this falls beyond the scope of this initial study. Future research could (and should) indeed investigate a range of EP parameters to build upon our foundational findings and further refine the application of EP in gene therapy.


7.How did you calculate the weighted average fluorescence intensity? Please interpret your formula.

Response:

The weighted average was calculated as follows:, where M was the number of distinct values, Ii was the intensity value, ni was the frequency of Ii, and N was the total number of pixels in the image.

This approach allowed us to characterize each fluorescence image with a single intensity value that represented the nominal transduction efficiency observed across that image, allowing comparison with other images from other treatment conditions.


8.The mechanism of the enhanced transduction in molecular level need further research, and you can have a brief discussion on it.

Response:

Please see our response to comment 4 above.


9.Line 309, “enhanced transduction by over 40-fold” doesn’t make sense since the transduction efficiency may not be in proportion to the concentration of AAV8, so you can’t simply multiply these two numbers. Please give more evidence to support this idea.



Response:

We thank the reviewer for raising the question regarding the relationship between AAV dosage and transduction efficiency. In response, we have conducted additional experiments which have been detailed in the newly added Supporting Information section of our manuscript. These experiments demonstrate a linear relationship between AAV dosage and transduction efficiency with an R-squared value of 0.927. Based on this new evidence, we have concluded that EP has effectively reduced the AAV dosage required for achieving the same level of transduction efficiency by more than 50-fold.



Minor Concerns:
1.Peaks of the saturated value around 100 needs further research.

Response:

We thank the reviewer for highlighting the significance of the peaks around the saturated value observed in our study. While our current research lays the groundwork in this area, the detailed exploration of these saturation peaks would be a valuable addition to future studies. 


2.Line 349, please discuss the affects of impulses on the transduction more in detail.

Response:

In our manuscript, we highlighted our approach of using fewer pulses and lower field strengths compared to studies involving naked plasmids. This choice was driven by two primary considerations: minimizing potential cellular damage and achieving effective transduction with AAV.

We carefully selected our EP parameters to strike a balance between maximizing transduction efficiency and maintaining cell viability. The parameters were not only tailored to reduce the likelihood of cellular stress or damage but also to ensure they were sufficient for effective AAV transduction. Our results demonstrate that these parameters, though less intensive than those used in some other studies, were adequately effective for the transduction by AAV of the targeted cells.

Attachment

Submitted filename: Response to Reviewers.docx

pone.0298866.s003.docx (49KB, docx)

Decision Letter 1

Chen Ling

1 Feb 2024

Electric Pulse Exposure Reduces AAV8 Dosage Required to Transduce HepG2 Cells

PONE-D-23-36580R1

Dear Dr. Yao,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Chen Ling, Ph.D.

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Overall, the author responded well to the questions raised by several reviewers and made modifications to the title and some parts of the content, positioning the scope of the article as AAV8 and HepG2 cell line derived from hepatocyte. At the same time, it is also suggested that this significant improvement in efficiency may be caused by changes in cell membrane pores caused by electrical pulses.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Negin P. Martin

Reviewer #2: Yes: Chen Ling

**********

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Checklist. Human participants research checklist.

    (DOCX)

    pone.0298866.s001.docx (52.7KB, docx)
    S1 Fig. Verification of the liner relationship between AAV dosage and fluorescence intensity.

    To substantiate the conclusions drawn in our main text regarding the impact of EP on transduction efficiency, we conducted a set of controlled experiments. The objective was to quantitatively establish the relationship between AAV dosage and the resultant transduction efficiency within HepG2. The experiments involved the application of three distinct dosages of AAV: 0.25×105, 1.75×105, and 2.5×105 vg/cell. Post-transduction, the cells were imaged and analyzed to obtain fluorescence intensity measurements, which served as a proxy for transduction efficiency. These intensity values were then normalized and rescaled to create a standard range from 0 to 100, facilitating comparison across varying AAV dosages. A linear regression analysis was performed on the weighted average fluorescence intensities against the corresponding AAV dosages. This analysis yielded an R-squared value of 0.927, indicating a linear relationship between AAV dosage and transduction efficiency.

    (TIF)

    pone.0298866.s002.tif (113.4KB, tif)
    Attachment

    Submitted filename: Response to Reviewers.docx

    pone.0298866.s003.docx (49KB, docx)

    Data Availability Statement

    All the raw data needed to replicate the results of the study are included as either Supporting Information or uploaded to the Open Science Framework. The URL link to the data in the Open Science Framework is https://osf.io/7xy8n/.

    Articles from PLOS ONE are provided here courtesy of PLOS

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