## 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_.