Open AccessArticle

Localized In Vivo Electro Gene Therapy (LiveGT)-Mediated Skeletal Muscle Protein Factory Reprogramming

Jacob Hensley

¹,

Michael Francis

^2,3,4,

Alex Otten

^1,4

Nadezhda Korostyleva

¹,

Tina Gagliardo

¹ and

Anna Bulysheva

^1,4,*

Department of Medical Engineering, University of South Florida, Tampa, FL 33620, USA

Asante Bio, Tampa, FL 33612, USA

Department of Orthopaedics and Sports Medicine, University of South Florida, Tampa, FL 33620, USA

⁴

Pele Therapeutics, Tampa, FL 33612, USA

Author to whom correspondence should be addressed.

Appl. Sci. 2024, 14(23), 11298; https://doi.org/10.3390/app142311298

Submission received: 1 October 2024 / Revised: 21 November 2024 / Accepted: 23 November 2024 / Published: 4 December 2024

(This article belongs to the Special Issue Applications of Pulsed Electric Field (PEF) Interactions with Biological Cells)

Download

Browse Figures

Figure 1
The in vitro pulse parameter is illustrated with a broken axis, along with 6 monophasic pulses with a length of 100 μs and a 250ms interval (A). A single representative waveform was recorded by an oscilloscope to ensure a square waveform with the correct voltage and pulse length was induced (B). "> Figure 2
Waveforms for in vivo reporter gene delivery. The first in vivo group was pulsed with 90 V of 0.5 µs duration followed by a 45 V pulse with 1 µs duration, which were repeated to yield a biphasic pulse wave (A). The second group was transfected using a 90 V pulse with 1 µs length followed by a 45 V pulse with 2 µs length (B). The last biphasic group was pulsed 50 V of 1 µs duration followed by a 25 V of 2 µs duration (C). Another in vivo group was pulsed with 8 monophasic pulses of 90 V each lasting 150 ms (D). "> Figure 3
GET significantly enhances pDNA delivery. Expression of human insulin (A), human glucokinase (B), insulin and glucokinase (GCK) on the same plasmid (SP) (C), and insulin and glucokinase on different plasmid (DP) backbones (D). Transfection efficiency calculated from three fields of view was significantly higher in all pulsed groups than in the non-pulsed controls (E). High co-localization of insulin and glucokinase was observed for both co-delivery conditions (F). *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001. "> Figure 4
Monophasic GET co-delivery of insulin and glucokinase significantly increases cellular glucose consumption. Glucose consumption over six days compared to glucose metabolism in controls (A). The highest levels of media glucose depletion are seen on day three (B). ***: p ≤ 0.001; ****: p ≤ 0.0001. "> Figure 5
GET significantly enhances insulin production and release into media. Protein expression in the same plasmid (SP) group in both high-glucose (A) and low-glucose (B) (Glc) conditions and different plasmid (DP) groups in high-glucose media (D) and low-glucose media (E) when compared to high-glucose (G) and low-glucose (H) controls. Protein expression was measured using immunofluorescence microscopy (C). Insulin expression was significantly enhanced in media as measured via ELISA in high-glucose (F) and low-glucose (I) conditions. ***: p ≤ 0.001; ****: p ≤ 0.0001. "> Figure 6
Glucokinase acts as a glucose sensor, preventing hypoglycemia (A). Change in glucose consumption based on cells in high- and low-glucose conditions when transfected with pDNA (B,C); *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001. "> Figure 7
LiveGT significantly enhances luciferase-encoding gene delivery and expression in skeletal muscle over 6 months. Monopolar, monophasic pulses resulted in the highest expression (>100 fold increase over IO; p < 0.05). "> Figure 8
Insulin and glucokinase liveGT increases serum human insulin levels while reducing serum glucose levels. Exogenous (human) insulin levels in serum were significantly elevated over 21 days (A) (p < 0.01). Fed serum glucose levels were significantly lower with insulin and glucokinase co-delivery via liveGT (B) (p < 0.05). Animals maintained healthy weight gain over 10 months (C). LiveGT had no deleterious effect on survival according to Kaplan–Meier survival analysis (D). ">

Versions Notes

Abstract

Gene electrotransfer (GET) has gained significant momentum as a non-viral gene delivery method for various clinical applications, primarily in the cancer immunotherapy and vaccine development space. Preclinical studies have demonstrated exogenous gene delivery and expression in various tissues, including the liver, skin, cardiac muscle, and skeletal muscle. However, protein replacement applications of this technology have yet to be fully actuated. Plasmid DNA skeletal muscle delivery has been shown to maintain expression for up to 18 months. In the current study, we evaluated localized skeletal muscle delivery for protein replacement applications. We developed localized in vivo electro gene therapy (liveGT) protocols utilizing mono- and biphasic pulse sequences for localized pulse delivery directly to skeletal muscle with a custom monopolar platinum electrode. Plasmid DNA encoding human insulin and human glucokinase were chosen for this study to evaluate the liveGT platform for protein replacement potential. Initial in vitro GET was performed in mouse myoblasts to evaluate human insulin and glucokinase co-delivery. This was followed by liveGT-mediated reporter gene delivery in the skeletal muscle of Sprague–Dawley rats for pulse sequence selection. Protein replacement potential was evaluated in healthy (non-diabetic) rats with liveGT-mediated human insulin and glucokinase co-delivery to skeletal muscle. Human and rat insulin levels were measured via ELISA over the course of 3 months. Fed-state blood glucose measurements were monitored in correlation with serum human insulin levels. LiveGT-mediated skeletal muscle reprogramming successfully produced physiological levels of human insulin in serum over the course of 3 months. Hypo- and hyperglycemic events were not observed. Therefore, liveGT is a safe and viable platform for potential protein replacement therapies.

Keywords:

insulin; gene electrotransfer; liveGT; gene therapy; glucose; protein replacement therapy

1. Introduction

Gene electrotransfer (GET) is a non-viral gene delivery method that utilizes a short, pulsed electric field to transfect polar molecules such as plasmid DNA (pDNA) into cells through the cell membrane to enhance gene expression [1]. In vivo gene delivery via GET was first reported in 1996 in a rat liver utilizing reporter gene luciferase [2]. Monophasic pulse shapes of millisecond-scale durations were used in this first in vivo work and are still known to achieve transfection and expression patterns [2,3]. Significant progress has been achieved with classic GET protocols, including groundbreaking work in cancer immunotherapy and vaccine development [4,5,6]. However, the translation of GET protocols to protein replacement therapies remains elusive. Important challenges remain and include difficulties in reaching appropriate physiological levels of specific protein expression for a therapeutically meaningful duration of time. Other gene delivery methods such as viral based methods are known to achieve higher levels of expression and longer durations of expression than are typically possible with traditional GET with pDNA delivery [7,8]. However, GET-mediated naked pDNA delivery does have advantages of lower risk of immunogenicity and integration compared to viral methods [8]. The present study focused on establishing the feasibility of using gene electrotransfer as an alternative for protein replacement therapies and testing whether physiologically relevant protein levels could be obtained and maintained for an extended period of several months with reporters and a representative effector protein.

While delivery with reporter genes encoding firefly luciferase is common in evaluating deliver efficiency, they do not directly translate into known protein levels of effector protein. Therefore, in this study we chose to work directly with a potential therapeutic gene encoding human insulin to evaluate pulsing parameters and the resulting circulating blood levels of insulin. Insulin is the first FDA-approved recombinant protein for replacement therapy and is arguably one of the best studied [9,10]. Thus, it was selected as our target protein of choice. In a previous study, an adeno-associated viral (AAV) vector containing serotype 1 encoding for insulin and glucokinase in diabetic dogs resulted in the normalization of fasting blood glucose levels and accelerated disposal of glucose after feeding [11]; therefore, we opted to focus on the co-delivery of insulin and glucokinase encoding pDNA with liveGT.

Skeletal muscle was selected as the target tissue for multiple reasons. Skeletal muscle is considered to be an endocrine organ, with an endogenous ability to secrete many proteins called myokines into the bloodstream [12]. Additionally, due to the low rate of cell turnover, any pDNA that is introduced into myocytes is known to be expressed for many months—sometimes up to 18 months [13,14]. In contrast, other cell types like epithelial cells may turn over in just a few days [15]. Other cells that turn over quicker also quickly lose their ability produce exogenous protein, as non-integrating pDNA is cleared out of the cells during cell division [14]. Therefore, we consider skeletal muscle a perfect target for reprogramming into replacement protein factories for blood-circulating proteins such as insulin. Additionally, specifically in the case of insulin replacement therapy development, it is important that skeletal muscle cells endogenously express glucose transporter-4 (GLUT4) [16]. GLUT4 functions to transport glucose across the plasma membrane but only in the presence of insulin [16]. Therefore, GLUT4 is hypothesized to act as a glucose sensor, together with exogenously delivered glucokinase. Combined, these qualities make skeletal muscle the perfect platform for reprogramming into an in vivo protein factory for protein replacement therapies with liveGT.

Overall, our study aimed to evaluate transfection efficiency using both monophasic and biphasic waveforms and to examine the functionality of transfected proteins for potential prolonged protein replacement therapy. We employed GET to enhance gene expression in C2C12 myoblasts in vitro and developed new liveGT protocols in rat skeletal muscle in vivo. In these experiments, we not only measured expression but also confirmed insulin function in in vitro and in vivo settings by measuring media and blood glucose levels in an insulin-dependent manner by transiently transfecting Nanoplasmid^® DNA encoding insulin and glucokinase. Achieving prolonged co-expression of insulin and glucokinase, along with functionally decreasing blood glucose levels, offers promising prospects for the treatment of various rare disorders that require continuous recombinant protein therapies while avoiding common drawbacks associated with viral delivery approaches.

2. Materials and Methods

2.1. Plasmids

Nanoplasmid^® DNAs used for glucose modulation include NTC9835-Insulin-DDK-BGH, NTC9835-Glucokinase-DDK-BGH, and NTC9835-Insulin-Myc-Glucokinase-DDK (Nature Technologies Corporation, Lincoln, NE, USA). These plasmids encode for full-length human insulin and glucokinase, respectively. Nanoplasmid^®-encoding firefly luciferase, NTC9835-luciferase-DDK-BGH, was utilized for in vivo reporter gene experiments. Plasmid DNA was suspended in sterile water at 2 mg/mL. Endotoxin analysis was performed by Nature Technologies Corporation, with a provided certificate indicating levels below significance.

2.2. Cells and Mitomycin C Treatment

For the in vitro investigation of pulsing parameters, we used C2C12 mouse myoblasts obtained through a generous donation from Dr. Akintewe. The cells were purchased from the ATCC organization under number CRL-1722. The cell cycle was arrested with mitomycin C treatment to inhibit cell proliferation and allow for longer pDNA expression. Cells were incubated in 10 mL of 10 µg/mL of mitomycin C for 3 h.

2.3. Electroporation Buffer

Myoblasts were resuspended in an electroporation buffer to ensure square wave pulses could be delivered to the cells by increasing the impedance of the solution. The buffer contained 1 mM MgCl₂, 0.7 mM KCl, 10 mM HEPES, and 285 mM sucrose diluted in purified water [17].

2.4. GET Co-Delivery of pDNA Encoding Human Insulin and Glucokinase In Vitro

Initial gene electrotransfer experiments were performed in vitro, utilizing C2C12 myoblasts and a BTX ECM 830 Square Wave Electroporation System (BTX, Holliston, MA, USA), with a single pulse parameter and four experimental groups, along with two control groups. Based on previous studies [4], 6 monophasic pulses were applied at 130 V, each with a duration of 100 μs and an interval of 250 ms. The pulse length, voltage, and interval were measured with a TBS2000 Series Digital Oscilloscope (Tektronix, Beaverton, OR, USA) (Figure 1B).

Myoblasts were suspended in electroporation buffer, placed in a 1mm parallel plate cuvette, and plated at a density of 3.4 × 10³ cells/cm² in a 24-well plate. Nanoplasmid^® DNA encoding for the following groups was resuspended with each sample in the cuvette: 10 µg Insulin-DDK, 10 µg Glucokinase-DDK, 10 µg Insulin-DDK, and 10 µg Glucokinase-DDK on separate plasmids, as well as 10 µg of pDNA encoding Insulin-myc-Glucokinase-DDK on the sample plasmid. The two control groups included myoblasts with 10 µg of Nanoplasmid^® encoding for Insulin-myc-Glucokinase-DDK and no pulses or myoblasts with no pDNA. Following thorough resuspension, cuvettes were pulsed and allowed 5 min undisturbed to ensure full membrane recovery. The groups were then plated in 1 mL of high-glucose Dulbecco’s Modified Eagle medium (DMEM), 10% fetal bovine serum, 1% antibiotic antimitotic solution, L-Glutamine, and sodium pyruvate. The effects of co-delivery of insulin and glucokinase were also observed in the low- and high-glucose media conditions to model transfected cells’ response to environmental variability. The glucose concentration of the high-glucose complete media was 400 mg/dL, while the concentration of low-glucose media was 100 mg/dL.

Following GET, media glucose levels of each group were measured and normalized to complete media that were not exposed to cells each day for seven days using GlucCellTM^® (Esco VacciXcell, Singapore). An ultrasensitive human insulin ELISA (Alpco, Macedon, NY, USA) was used according to manufacturer instructions to measure the secretion of insulin into the media.

2.5. Immunofluorescence Staining and Fluorescence Microscopy

Following day-seven glucose measurements, all groups were washed with 1× phosphate-buffered saline and fixed with 4% paraformaldehyde (PFA) for 30 min at 4 °C. The plate was washed with 1× phosphate-buffered saline tween (PBST), permeabilized using 0.25% TritonX for 20 min, blocked using 4% Bovine Serum Albumin (BSA) in 1× PBST, placed on a shaker at room temperature for 60 min, and incubated with either anti-insulin antibody and/or anti-DDK antibody 1:200 in 4% BSA overnight at 4 °C. The plate was then washed with 1× PBST 5 times, blocked for 60 min in 4% BSA, and incubated with anti-rat IgG 488 or anti-mouse IgG 546 1:200 in 4% BSA and counterstained with spectral DAPI for 1 h on a shaker at room temperature. The plate was then washed 5 times with PBST to remove excess antibody and DAPI.

Fluorescence microscopy was performed immediately following PBST washes using ECHO Revolution (ECHO, San Diego, CA, USA). Images were taken in a 10× objective with a DAPI, FITC, and Tex Red multichannel overlay. Myoblasts were then analyzed by counting the percent of cells expressing the protein of interest from three random fields of view per well (n = 12).

2.6. Animals

Non-diabetic Sprague–Dawley male rats were purchased and acclimated for a seven-day period before any procedures. All experiments utilizing animals followed an approved Institutional Animal Care and Use Committee protocol (#IS00011369) at an AAALAC-accredited facility at the University of South Florida.

2.7. In Vivo Reporter Gene Delivery via liveGT

The Nanoplasmid^® DNA encoding firefly luciferase served as the reporter gene, facilitating the measurement of bioluminescence and protein expression. Rats were anesthetized with 2–3% Isoflurane. Once anesthetized, bilateral shaving of the rats’ sides was performed, followed by the application of ultrasound gel to one side. The rats were then positioned on a grounding plate, and four incisions were made on each flank to expose the skeletal muscle. Intramuscular injections of 50 µL (2 µg/µL) Nanoplasmid^® DNA encoding luciferase were administered at an angled position with a 25-gauge needle.

A 1 cm² square monopolar platinum electrode designed for a more uniform electric field and increased gene delivery aligned with the grounding plate was placed in direct contact with the rat’s skeletal muscle, and specific pulsing conditions were applied [18]. These liveGT conditions included asymmetric biphasic pulses, as well as monophasic pulses, as can be seen in Figure 2. The first group received 32 trains of 32,367 pulses at 90 V/45 V with alternating 0.5 µs/1 µs pulse lengths (Figure 2C). The second group received 8 trains of 32,367 pulses at 90 V/45 V with 1 µs/2 µs pulse lengths (Figure 2B). The third group—identical to the first group in pulse length, number of pulses, and number of train pulses—was administered at an applied voltage of 50 V/25 V (Figure 2A). Additionally, a monophasic condition was applied, consisting of square wave pulses with 8 pulses of 150 ms each at an applied voltage of 90 V (Figure 2D). This pulsing parameter was sourced from previous work yielding high myocardial gene expression [19]. All experimental conditions were compared against injection-only sites that received pDNA and did not undergo pulsing. Following completion of pulsing or injection, the incisions were closed.

2.8. Bioluminescence Measurements

Bioluminescence quantification was conducted utilizing the In vivo Imaging System (IVIS, PerkinElmer, Waltham, MA, USA). Rats underwent initial anesthesia using 2–3% isoflurane. Subsequently, each rat received a subcutaneous injection of D-luciferin at a standard concentration of 150 mg/kg of body weight, as per manufacturer instructions [20]. Four regions of interest (ROIs), each with a diameter of 1 cm, were defined for bioluminescence measurement, expressed as a flux in photons per second (p/s). The flux within each ROI was then normalized by subtracting the flux from a non-ROI background area. Bioluminescence from the rat’s flank was measured at 5 min intervals over a duration of 30 min to ascertain peak bioluminescence values. Bioluminescence was measured on days 1, 2, 7, 21, 29, 91, and 182 to measure the level of prolonged expression of a transient transfection to rat skeletal muscle cells.

2.9. LiveGT of Insulin and Glucokinase

Following analysis of the reporter gene experiments, a monophasic pulse was selected for further in vivo investigations. Rats were anesthetized with 2–3% isoflurane and shaved on both flanks. Conducting gel was applied to one flank, and the rat was positioned on the grounding electrode. An incision was made on the rat’s bicep femoris, exposing the skeletal muscle. Upon exposure, intramuscular injections of 50 µL (2 µg/µL) Nanoplasmid^® DNA encoding human insulin and glucokinase were administered. The platinum electrode was then applied, and the monophasic pulsing condition was employed. Following pulsing, the incision was sutured, and the same procedure was repeated on the contralateral flank. Thus, each of the four rats (n = 4) received pulsing at two sites, and the rats were weighed daily to ensure no significant decrease in body mass.

Following the transfection, 600 µL of blood was drawn from the rats on days 2, 7, 14, 21, and 91. Serum was extracted from the blood samples using BD Microtainer^® tubes (Lakes, NJ, USA) containing a clot-activator SST gel. Subsequently, the blood was centrifuged at 4 °C for 10 min at 2000× g to facilitate serum separation. Serum glucose levels were determined using blood glucose test strips (Ascensia Diabetes Care, Parsippany, NJ, USA). Additionally, the serum was analyzed using an Ultrasensitive Human Insulin ELISA kit (Alpco, Macedon, NY, USA) according to the manufacturer’s instructions.

2.10. Statistical Analysis

All quantitative data were compared using a two-way ANOVA and Tukey’s multiple comparison test, with p < 0.05 considered statistically significant.

3. Results

3.1. GET Enhances pDNA Delivery In Vitro

Transfection efficiency was assessed via immunofluorescence microscopy (Figure 3A–D). Compared to myoblasts receiving pDNA alone, all pulsed groups exhibited significant increases in protein expression of both insulin and glucokinase (Figure 3E) (p < 0.01). No significant differences in protein expression were observed among the pulsed groups (Figure 3E). Co-localization of insulin and glucokinase was higher in the group receiving a plasmid encoding both insulin and glucokinase on a single plasmid than in the group receiving separate plasmids encoding these proteins (Figure 3F).

3.2. Co-Expression of Insulin and Glucokinase Increases Glucose Consumption In Vitro

Following transfection with pDNA encoding insulin, glucokinase, or both, functional evaluation was conducted via media glucose consumption analysis. Over a span of 6 days, increased rates of glucose consumption were detected in both the same plasmid and different plasmid groups relative to the baseline glucose consumption of normal myoblasts (Figure 4A). A detailed examination of peak protein expression revealed notable enhancements in glucose consumption on the third day, which were evident across both the same plasmid and different plasmid groups, as well as the glucokinase group (Figure 4B). Insulin expression alone was not enough to significantly elevate glucose depletion compared to myocyte basal-level glucose metabolism.

3.3. Insulin and Glucokinase Co-Expression in High- vs. Low-Glucose Media

Protein expression levels were determined via immunofluorescence microscopy for both high-glucose (Figure 5A,D,G) and low-glucose (Figure 5B,E,H) conditions. Enhanced transfection efficiency was evident in groups subjected to pulsing compared to myoblasts receiving DNA alone (Figure 5C). Notably, no significant disparities were observed in transfection efficiency between low- and high-glucose media; however, slightly reduced protein expression was measured in the low-glucose media (Figure 5C). Detection of human insulin in the media was consistent across all groups transfected with DNA encoding insulin under both high-glucose (Figure 5F) and low-glucose (Figure 5I) conditions. Myoblasts that did not receive pDNA encoding insulin exhibited human insulin levels below the detection limit of the human insulin ELISA (Figure 5F,I).

3.4. Glucokinase Co-Expression Prevents Hypoglycemia

Following co-transfection with both insulin- and glucokinase-encoding pDNA on the same and different plasmids under high- and low-glucose media conditions, co-delivery significantly reduced media glucose in the high-glucose environment. Both the same and different plasmid groups exhibited decreases in media glucose levels ranging from 30 to 40 mg/dL under high-glucose conditions (Figure 6A–C). However, the change in glucose levels in low-glucose media ranged from 10 to 20 mg/dL (Figure 6A–C). Notably, under low-glucose conditions, media glucose levels remained above hypoglycemic thresholds (Figure 6A).

3.5. Prolonged Reporter Gene Expression In Vivo

In vivo bioluminescence assessments revealed a >100-fold significant increase in luciferase protein expression over injection only in a span of three months, with sustained elevated levels of luciferase expression persisting even six months following a single treatment with the monophasic pulsing condition (Figure 7). Conversely, only the 50 V/25 V biphasic condition significantly increased expression by up to 10-fold over injection only (Figure 7).

3.6. LiveGT of Insulin and Glucokinase Decreases Serum Glucose Levels In Vivo

Following increases in reporter gene luciferase transfection using the monophasic pulsing regimen, the same pulsing protocol was employed with Nanoplasmid^® DNA encoding both insulin and glucokinase. Analysis via human insulin ELISA demonstrated significant increases in serum human insulin levels persisting for up to 3 months (Figure 8A). Additionally, reductions in glucose levels relative to controls were noted on days 7 and 14 (Figure 8B). Normal rat growth was observed over time (Figure 8C), with all rats gaining proper weight during observation time post treatment. According to a Kaplan–Meier survival plot, liveGT delivery is safe, as survival was the same between treated and untreated animals (Figure 8D).

4. Discussion

Results of the in vitro experiments demonstrate that utilizing GET as a vehicle for the delivery of pDNA significantly increases protein expression of insulin alone and glucokinase alone, as well as the co-expression of insulin and glucokinase (Figure 3). Additionally, delivery of pDNA encoding full-length human insulin and co-delivery of insulin and glucokinase significantly increase glucose metabolism compared to basal-cell metabolism of glucose. (Figure 4). This indicates that GET-mediated gene delivery allows for the expression of functional therapeutic proteins, as demonstrated by the modulation of glucose concentration in media.

To understand the functional role of glucokinase as a sensor and glucose uptake and metabolism within skeletal myoblast cells, an experimental design aimed to assess glucose utilization under both elevated and reduced glucose concentrations. Type 1 Diabetes (T1D) often leads to hyperglycemic conditions; however, between meals, blood glucose levels of patients with and without T1D differ by around 20% [21]. Too much insulin administration can lead to hypoglycemia, which can be potentially dangerous to the patient. Therefore, it is important to implement a strategy that accounts for both hyper- and hypoglycemia. Specifically, high-glucose (400 mg/dL) and low-glucose (100 mg/dL) environments were employed to gauge variability in glucose levels following the in vitro introduction of exogenous insulin and glucokinase via GET. Results indicate that in low-glucose conditions, myoblasts consumed less glucose, keeping the environment above hypoglycemic levels (Figure 6). In high-glucose conditions, glucose consumption was elevated, significantly reducing hyperglycemia (Figure 6). This is a significant finding, particularly for T1D insulin protein replacement applications, and it is consistent with our hypothesis that the co-delivery of insulin and glucokinase may lead to the modulation of blood glucose within the safe range. These in vitro results led to further in vivo investigation.

In vivo delivery presents several challenges that were addressed in this study. Our previous studies indicated that gene delivery was highly improved in skin and cardiac muscle tissues using monophasic pulses and a monopolar electrode [18,19]. Therefore, our initial in vivo experiments focused on adapting pulsing parameters for direct delivery to skeletal muscles with a monopolar electrode (Figure 7). An in vivo non-diabetic rat model was employed to assess expression levels of firefly luciferase reporter in skeletal muscle. Utilizing our monopolar, platinum electrode, the cell membrane of skeletal muscle cells was transiently permeabilized with monophasic and biphasic pulsing parameters. Monophasic parameters resulted in overwhelmingly higher expression levels than any other conditions, despite significant skeletal muscle contractions when applied without nerve-blocking agents (Figure 7). Remarkably, this enhanced transfection efficiency was sustained, with continued elevated protein expression observed at the 6-month mark (Figure 7). The sustained expression of protein holds significant importance for the development of long-term therapeutic strategies targeting various protein-based therapies [13]. Selected biphasic pulses resulted in mild enhancement of expression and were not selected for further evaluation of therapeutic gene delivery.

Due to the notable increase in protein expression observed under the monophasic condition (Figure 7), rats were subjected to transfection with Nanoplasmid^® DNA encoding both human insulin and human glucokinase using 90 V, 150 ms monophasic pulsing parameters. The outcomes of liveGT conducted in vivo revealed a sustained release of functional human insulin into the bloodstream, as evidenced by Ultrasensitive Human Insulin ELISA measurements (Figure 8A), without cross-reactivity with rat insulin. It is important to note that myotubule secretion of insulin into the bloodstream is essential for insulin receptor binding, facilitating the translocation of GLUT-4 to the membrane for glucose uptake.

Furthermore, our study demonstrated not only the release but also the functional activity of the insulin released into the bloodstream, as evidenced by a significant reduction in rat glucose levels over a two-week period (Figure 8B) during which physiological levels of human insulin were detected. A Kaplan–Meier survival plot illustrated no significant disparities in survival between the liveGT groups and untreated control animals (Figure 8D). These findings illustrate the sustained secretion of proteins within skeletal muscle cells for prolonged therapeutic efficacy. Additionally, the results support the proof-of-concept idea that functionally competent protein expressed in the physiological range can be delivered with liveGT-enhanced pDNA delivery of insulin and glucokinase. Additionally, co-expression of insulin and glucokinase retains the capacity to effectively reduce blood glucose levels in an insulin-dependent manner. These findings collectively highlight the promising potential of this approach for sustained therapeutic interventions.

5. Conclusions

Experiments were designed to measure the effectiveness of liveGT for long-term protein secretion and functionality. In vitro GET of pDNA encoding insulin and glucokinase resulted in significantly increased protein expression and glucose consumption. Glucokinase demonstrated its ability to act as a sensor, preventing hypoglycemia. Delivery of reporter genes using localized in vivo electro gene therapy (liveGT) with biphasic pulses may reduce muscle twitching and increase delivery efficiency, though further optimization is needed to achieve physiologically relevant expression levels. Moreover, liveGT of insulin and glucokinase with monophasic pulses led to significant decreases in serum glucose levels for up to 21 days and increased serum human insulin levels. Therefore, liveGT has the potential for prolonged protein expression, offering an alternative gene therapy platform for protein replacement therapies.

Author Contributions

Conceptualization, A.B. and M.F.; methodology, A.B., J.H. and M.F.; software, A.O.; validation, J.H. and A.B.; formal analysis, J.H. and A.B.; investigation, J.H., N.K., T.G. and A.B.; resources, A.B.; data curation, J.H., N.K., A.O., T.G. and A.B.; writing—original draft preparation, J.H., N.K. and A.B.; writing—review and editing, J.H., N.K., M.F. and A.B.; visualization, J.H. and A.B.; supervision, A.B.; project administration, A.B.; funding acquisition, A.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded, in part, by the Commonwealth Health Research Board (grant number 236-13-22) and start-up funds from the University of South Florida.

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Animal Care and Use Committee at the University of South Florida (IS0011369; approved 13 January 2023).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Acknowledgments

The authors would like to thank Mark Jaroszeski allowing the utilization of his lab space to conduct in vitro experiments.

Conflicts of Interest

Author Michael Francis was employed by the company Asante Bio and Pele Therapeutics. Authors Alex Otten and Anna Bulysheva were employed by the company Pele Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

Bureau, M.F.; Gehl, J.; Deleuze, V.; Mir, L.M.; Scherman, D. Importance of Association between Permeabilization and Electrophoretic Forces for Intramuscular DNA Electrotransfer. Biochim. Biophys. Acta Gen. Subj. 2000, 1474, 353–359. [Google Scholar] [CrossRef] [PubMed]
Heller, R.; Jaroszeski, M.; Atkin, A.; Moradpour, D.; Gilbert, R.; Wands, J.; Nicolau, C. In Vivo Gene Electroinjection and Expression in Rat Liver. FEBS Lett. 1996, 389, 225–228. [Google Scholar] [CrossRef] [PubMed]
Potočnik, T.; Lebar, A.M.; Kos, Š.; Reberšek, M.; Pirc, E.; Serša, G.; Miklavčič, D. Effect of Experimental Electrical and Biological Parameters on Gene Transfer by Electroporation: A Systematic Review and Meta-Analysis. Pharmaceutics 2022, 14, 2700. [Google Scholar] [CrossRef] [PubMed]
Daud, A.I.; DeConti, R.C.; Andrews, S.; Urbas, P.; Riker, A.I.; Sondak, V.K.; Munster, P.N.; Sullivan, D.M.; Ugen, K.E.; Messina, J.L.; et al. Phase I Trial of Interleukin-12 Plasmid Electroporation in Patients with Metastatic Melanoma. J. Clin. Oncol. 2008, 26, 5896–5903. [Google Scholar] [CrossRef] [PubMed]
Kisakov, D.N.; Belyakov, I.M.; Kisakova, L.A.; Yakovlev, V.A.; Tigeeva, E.V.; Karpenko, L.I. The Use of Electroporation to Deliver DNA-Based Vaccines. Expert Rev. Vaccines 2023, 23, 102–123. [Google Scholar] [CrossRef] [PubMed]
Bodles-Brakhop, A.M.; Heller, R.; Draghia-Akli, R. Electroporation for the Delivery of DNA-Based Vaccines and Immunotherapeutics: Current Clinical Developments. Mol. Ther. 2009, 17, 585–592. [Google Scholar] [CrossRef] [PubMed]
Djurovic, S.; Iversen, N.; Jeansson, S.; Hoover, F.; Christensen, G. Comparison of Nonviral Transfection and Adeno-Associated Viral Transduction on Cardiomyocytes. Mol. Biotechnol. 2004, 28, 21–32. [Google Scholar] [CrossRef] [PubMed]
Young, J.L.; Benoit, J.N.; Dean, D.A. Effect of a DNA Nuclear Targeting Sequence on Gene Transfer and Expression of Plasmids in the Intact Vasculature. Gene Ther. 2003, 10, 1465–1470. [Google Scholar] [CrossRef] [PubMed]
Mathieu, C.; Martens, P.-J.; Vangoitsenhoven, R. One Hundred Years of Insulin Therapy. Nat. Rev. Endocrinol. 2021, 17, 715–725. [Google Scholar] [CrossRef] [PubMed]
Fosgerau, K.; Hoffmann, T. Peptide Therapeutics: Current Status and Future Directions. Drug Discov. Today 2014, 20, 122–128. [Google Scholar] [CrossRef] [PubMed]
Jaén, M.L.; Vilà, L.; Elias, I.; Jimenez, V.; Rodó, J.; Maggioni, L.; Gopegui, R.R.-D.; Garcia, M.; Muñoz, S.; Callejas, D.; et al. Long-Term Efficacy and Safety of Insulin and Glucokinase Gene Therapy for Diabetes: 8-Year Follow-Up in Dogs. Mol. Ther. Methods Clin. Dev. 2017, 6, 1–7. [Google Scholar] [CrossRef] [PubMed]
Hoffmann, C.; Weigert, C. Skeletal Muscle as an Endocrine Organ: The Role of Myokines in Exercise Adaptations. Cold Spring Harb. Perspect. Med. 2017, 7, a029793. [Google Scholar] [CrossRef] [PubMed]
Mir, L.M.; Bureau, M.F.; Gehl, J.; Rangara, R.; Rouy, D.; Caillaud, J.-M.; Delaere, P.; Branellec, D.; Schwartz, B.; Scherman, D. High-Efficiency Gene Transfer into Skeletal Muscle Mediated by Electric Pulses. Proc. Natl. Acad. Sci. USA 1999, 96, 4262–4267. [Google Scholar] [CrossRef] [PubMed]
McMahon, J.M.; Wells, D.J. Electroporation for Gene Transfer to Skeletal Muscles. BioDrugs 2004, 18, 155–165. [Google Scholar] [CrossRef] [PubMed]
Arike, L.; Seiman, A.; Van Der Post, S.; Piñeiro, A.M.R.; Ermund, A.; Schütte, A.; Bäckhed, F.; Johansson, M.E.V.; Hansson, G.C. Protein Turnover in Epithelial Cells and Mucus along the Gastrointestinal Tract Is Coordinated by the Spatial Location and Microbiota. Cell Rep. 2020, 30, 1077–1087.e3. [Google Scholar] [CrossRef] [PubMed]
Stöckli, J.; Fazakerley, D.J.; James, D.E. GLUT4 Exocytosis. J. Cell Sci. 2011, 124, 4147–4159. [Google Scholar] [CrossRef] [PubMed]
Sherba, J.J.; Hogquist, S.; Lin, H.; Shan, J.W.; Shreiber, D.I.; Zahn, J.D. The Effects of Electroporation Buffer Composition on Cell Viability and Electro-Transfection Efficiency. Sci. Rep. 2020, 10, 3053. [Google Scholar] [CrossRef] [PubMed]
Bulysheva, A.; Heller, L.; Francis, M.; Varghese, F.; Boye, C.; Heller, R. Monopolar Gene Electrotransfer Enhances Plasmid DNA Delivery to Skin. Bioelectrochemistry 2021, 140, 107814. [Google Scholar] [CrossRef] [PubMed]
Boye, C.; Arpag, S.; Burcus, N.; Lundberg, C.; DeClemente, S.; Heller, R.; Francis, M.; Bulysheva, A. Cardioporation Enhances Myocardial Gene Expression in Rat Heart. Bioelectrochemistry 2021, 142, 107892. [Google Scholar] [CrossRef] [PubMed]
Gold Biotechnology. D-Luciferin In Vivo Protocol; Gold Biotechnology: St. Louis, MO, USA, 2013; Available online: http://www.goldbio.com/product/1078/d-luciferin-potassium-salt-proven-and-published (accessed on 2 November 2024).
Mathew, T.K.; Zubair, M.; Tadi, P. Blood Glucose Monitoring. Last Update: 23 April 2023. Available online: https://www.ncbi.nlm.nih.gov/books/NBK555976/ (accessed on 2 November 2024).

Figure 1. The in vitro pulse parameter is illustrated with a broken axis, along with 6 monophasic pulses with a length of 100 μs and a 250ms interval (A). A single representative waveform was recorded by an oscilloscope to ensure a square waveform with the correct voltage and pulse length was induced (B).

Figure 2. Waveforms for in vivo reporter gene delivery. The first in vivo group was pulsed with 90 V of 0.5 µs duration followed by a 45 V pulse with 1 µs duration, which were repeated to yield a biphasic pulse wave (A). The second group was transfected using a 90 V pulse with 1 µs length followed by a 45 V pulse with 2 µs length (B). The last biphasic group was pulsed 50 V of 1 µs duration followed by a 25 V of 2 µs duration (C). Another in vivo group was pulsed with 8 monophasic pulses of 90 V each lasting 150 ms (D).

Figure 3. GET significantly enhances pDNA delivery. Expression of human insulin (A), human glucokinase (B), insulin and glucokinase (GCK) on the same plasmid (SP) (C), and insulin and glucokinase on different plasmid (DP) backbones (D). Transfection efficiency calculated from three fields of view was significantly higher in all pulsed groups than in the non-pulsed controls (E). High co-localization of insulin and glucokinase was observed for both co-delivery conditions (F). *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001.

Figure 4. Monophasic GET co-delivery of insulin and glucokinase significantly increases cellular glucose consumption. Glucose consumption over six days compared to glucose metabolism in controls (A). The highest levels of media glucose depletion are seen on day three (B). ***: p ≤ 0.001; ****: p ≤ 0.0001.

Figure 5. GET significantly enhances insulin production and release into media. Protein expression in the same plasmid (SP) group in both high-glucose (A) and low-glucose (B) (Glc) conditions and different plasmid (DP) groups in high-glucose media (D) and low-glucose media (E) when compared to high-glucose (G) and low-glucose (H) controls. Protein expression was measured using immunofluorescence microscopy (C). Insulin expression was significantly enhanced in media as measured via ELISA in high-glucose (F) and low-glucose (I) conditions. ***: p ≤ 0.001; ****: p ≤ 0.0001.

Figure 6. Glucokinase acts as a glucose sensor, preventing hypoglycemia (A). Change in glucose consumption based on cells in high- and low-glucose conditions when transfected with pDNA (B,C); *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001.

Figure 7. LiveGT significantly enhances luciferase-encoding gene delivery and expression in skeletal muscle over 6 months. Monopolar, monophasic pulses resulted in the highest expression (>100 fold increase over IO; p < 0.05).

Figure 8. Insulin and glucokinase liveGT increases serum human insulin levels while reducing serum glucose levels. Exogenous (human) insulin levels in serum were significantly elevated over 21 days (A) (p < 0.01). Fed serum glucose levels were significantly lower with insulin and glucokinase co-delivery via liveGT (B) (p < 0.05). Animals maintained healthy weight gain over 10 months (C). LiveGT had no deleterious effect on survival according to Kaplan–Meier survival analysis (D).

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Share and Cite

MDPI and ACS Style

Hensley, J.; Francis, M.; Otten, A.; Korostyleva, N.; Gagliardo, T.; Bulysheva, A. Localized In Vivo Electro Gene Therapy (LiveGT)-Mediated Skeletal Muscle Protein Factory Reprogramming. Appl. Sci. 2024, 14, 11298. https://doi.org/10.3390/app142311298

AMA Style

Hensley J, Francis M, Otten A, Korostyleva N, Gagliardo T, Bulysheva A. Localized In Vivo Electro Gene Therapy (LiveGT)-Mediated Skeletal Muscle Protein Factory Reprogramming. Applied Sciences. 2024; 14(23):11298. https://doi.org/10.3390/app142311298

Chicago/Turabian Style

Hensley, Jacob, Michael Francis, Alex Otten, Nadezhda Korostyleva, Tina Gagliardo, and Anna Bulysheva. 2024. "Localized In Vivo Electro Gene Therapy (LiveGT)-Mediated Skeletal Muscle Protein Factory Reprogramming" Applied Sciences 14, no. 23: 11298. https://doi.org/10.3390/app142311298

APA Style

Hensley, J., Francis, M., Otten, A., Korostyleva, N., Gagliardo, T., & Bulysheva, A. (2024). Localized In Vivo Electro Gene Therapy (LiveGT)-Mediated Skeletal Muscle Protein Factory Reprogramming. Applied Sciences, 14(23), 11298. https://doi.org/10.3390/app142311298

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Menu