## 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 hyperglyic 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(r) 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.