Type 1 diabetes mellitus (T1DM) is an autoimmune disorder whereby the insulin-secreting β cells of the pancreas are destroyed. Under physiological conditions, β cells sense and respond to ever-changing blood glucose levels by secreting insulin, which acts on other tissues to promote glucose uptake from the blood and thus lower blood glucose levels. Without sufficient numbers of functional β cells, insulin production becomes inadequate and hyperglycemia ensues. Over time, chronic or poorly controlled hyperglycemia leads to numerous secondary complications, ultimately causing widespread tissue/organ damage and increased mortality. In fact, diabetes is the leading cause of end-stage kidney failure, retinopathy and associated blindness, non-traumatic amputations, and a variety of debilitating neuropathies. Despite active research, there is currently no cure for T1DM.

While there is currently no cure, several therapies exist to control blood glucose levels and limit adverse systemic damages resulting from sustained hyperglycemia. The most common form of treatment involves injection of synthetic insulin. Unfortunately, this option is cumbersome and unable to precisely control blood glucose levels. As a result, exogenous insulin therapy delays the onset and reduces the severity of secondary complications but is unable to prevent them [1] . In addition, exogenous insulin therapy is associated with hypoglycemia, which can lead to seizures, coma, or death. To better imitate normal physiology, specialized insulin pumps, dubbed artificial pancreata, have been developed that use a computer algorithm to deliver insulin [2] [3] or both insulin and glucagon [4] when needed by continuously monitoring blood glucose levels. Using this technology, better glycemic control can be attained compared to traditional insulin therapies. However, a variation in sensitivity with time and longevity of the implanted glucose sensor in vivo remain hurdles, as the glucose sensor will invariably accumulate serum or extracellular matrix proteins that compromise glucose measurements and subsequently affect the amount of insulin delivered. Ultimately, exogenous insulin therapy provides a suboptimal therapy for T1DM. In an effort to establish tighter control of blood glucose levels, whole-pancreas [5] or, to a lesser degree, pancreatic islet transplantation [6] surgeries have proven effective. However, pancreas transplantation therapy is severely hampered by a shortage of donor organs and further complicated by the need for lifelong immunosuppression. Transplantation of pancreatic islets was anticipated to minimize the impact of donor shortage, as islets from one donor could be expanded ex vivo to a quantity sufficient for multiple recipients. Unfortunately, equivalent successes like those observed with whole-pancreas transplantation have yet to be obtained [7] . Hence, there is clearly a need for alternative, state-of-the-art therapies. To that end, there have been considerable efforts to genetically engineer surrogate β cells to replace the function of those cells that were lost during the progression of T1DM.

In this review, we will begin by highlighting the unique features of β cells that make them so adept at controlling hyperglycemia. We will then discuss which of these features are absolutely critical to engineer a surrogate β cell capable of adequately controlling glycemia and describe the current efforts to genetically engineer surrogate β cells. Discussion on efforts to genetically engineer surrogate β cells will include insulin gene therapy―en- compassing various strategies to force the expression of insulin in non-β cells―and generation of β cells from both somatic and stem cells through expression of β cell-specific transcription factors. Along the way, we aim to point out advantages and disadvantages of each approach and ultimately propose the minimum requirements necessary for genetically engineering surrogate β cells with the potential to treat T1DM.

In order to engineer a surrogate β cell, it is important to understand the unique features of β cells that make them so adept at controlling glycemia and determine which of these features are absolutely critical to adequately perform its physiological function. The β cell possesses several remarkable features that together allow it to precisely control blood glucose levels. Specifically, the β cell has the ability to regulate insulin production at the transcriptional, post-transcriptional, translational, and post-translational levels, as well as the ability to store and secrete insulin in a highly regulated fashion in response to a variety of different stimuli, most notably glucose. While no individual feature is sufficient in itself to control blood glucose levels, nor specific to β cells, the combination of features makes it a remarkably sophisticated and competent cell for its task―one that has proven quite difficult to engineer identically.

First and foremost, the β cell is capable of sensing and responding to small changes in circulating glucose levels over a broad range of physiological concentrations (2 - 20 mM) through concentration-dependent entry and metabolism of glucose. They do so through expression glucose transporter-2 (GLUT2) and glucokinase. GLUT2 is a transmembrane carrier protein that enables bidirectional glucose transport across cell membranes, whereas glucokinase is an intracellular enzyme that initiates metabolism of glucose into glucose-6-phosphate. The critical feature of GLUT2 and glucokinase is their relatively high K_m for glucose (~17 and 8 mM, respectively), which allows their output to vary substantially across a broad range of glucose concentrations and thus serve as “glucose sensors” for β cells [8] . Without them, insulin secretion from β cells would be more like an “on-off switch” instead of a finely tuned trigger.

More remarkably, β cells have the ability to secrete insulin in a precisely regulated fashion in response to elevations in plasma glucose levels, as well as other secretagogues, to maintain euglycemia. β cells do so through their capacity to: 1) synthesize, process and store large quantities of insulin within secretory vesicles and 2) generate secondary stimulus-secretion coupling signals to activate rapid insulin vesicle exocytosis. It has been shown in rat β cells that >99% of all newly synthesized proinsulin molecules are packaged into secretory vesicles (see Figure 1), creating an enormous depot of insulin for rapid, on-demand secretion, which can occur within a minute after exposure to elevated glucose levels [9] . Further, β cells possess a unique combination of metabolic enzymes that ultimately leads to the generation of signals that adjust insulin secretion in response to glucose metabolism. β cells, but not other mammalian cell types, have negligible lactate dehydrogenase activity, so the pyruvate produced from glycolysis is not metabolized to lactate [10] . To compensate, β cells display increased pyruvate carboxylase activity to direct pyruvate almost entirely toward the mitochondria, where it is metabolized via the tricarboxylic acid cycle and oxidative phosphorylation. In so doing, β cells increase their ATP/ADP ratio and subsequently activate ATP-sensitive K⁺ channels, a key stimulus leading to insulin vesicle exocytosis. There is also a marked increase in mitochondrial metabolic shuttle activities to reoxidize cytosolic NADH to NAD⁺, a requirement normally provided by lactate dehydrogenase [11] . Increases in NADPH levels serve as another glucose-induced stimulus leading to insulin vesicle exocytosis [12] [13] . Together, the β cell has a unique balance of metabolic enzymes, structures, and mechanisms to generate the secondary stimulus-sec- retion coupling signals necessary for the almost instantaneous secretion of insulin upon exposure to secretagogues (see Figure 2 for schematic).

In addition to glucose, other circulating metabolites greatly influence β cell function. First, the gut produces peptide hormones, termed incretins, which serve as a line of communication between the gut and endocrine pancreas to augment post-prandial insulin production from β cells. These incretins, which include glucose-de- pendent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP1), bind specific receptors found predominantly on β cells to bolster insulin production [14] . Second, specific amino acids and free fatty acids can serve as insulin secretagogues. Although some amino acids and free fatty acids can, in certain combinations, actively promote insulin secretion, they have a more prominent role in amplifying the stimulatory effects of glucose [15] . Third, neuronal input can influence insulin secretion. In particular, metabolic stress can induce neuronal signals that affect insulin output [16] . Together, these inputs attenuate insulin output from β cells to more precisely regulate glucose levels.

Furthermore, β cells control insulin biosynthesis at the transcriptional, post-transcriptional, translational, and post-translational levels, with each level of control being regulated by glucose availability. β cells do not respond to glucose availability per se but to changes in the rate of glucose metabolism. At the transcriptional level, two transcription factors―PDX1 and MafA―have been identified as crucial regulators that promote insulin gene expression in response to altered glucose metabolism [17] [18] . Namely, it has been reported that PDX1 gets phosphorylated when the concentration of glucose is low, leading to its cytosolic localization [19] [20] . When glucose concentrations become elevated, PDX1 gets dephosphorylated and works cooperatively with MafA and BETA2 to promote insulin transcription [18] .

At the post-transcriptional and translational levels, β cells have glucose-dependent mechanisms to attenuate insulin mRNA stability and the rate of translation, respectively. Insulin mRNA has been estimated to be ~30% of the total mRNA within β cells, and its half-life can vary from anywhere between 29 to 77 hours depending on glucose availability [21] [22] . Association of an RNA-binding protein known as polypyrimidine tract-binding protein (PTB) with a pyrimidine-rich stretch in the 3’ UTR of the insulin mRNA has been shown to be responsible for glucose-dependent changes in its stability [23] [24] . However, overall changes in insulin production increase more dramatically due to glucose-induced changes in insulin mRNA translation. Wicksteed et al. found that glucose-stimulated increases in translation occur through the cooperative activities of a stem-loop at the 5’ UTR and the previously mentioned pyrimidine-rich stretch (UUGAA) in the 3’ UTR [24] . Sequences in the 5’ UTR have similarly been identified as important for glucose-induced increases in insulin mRNA translation [25] [26] , and protein-disulfide isomerase (PDI) has been identified as a binding protein responsible for glucose- stimulated insulin biosynthesis [27] .

Lastly, β cells possess specific post-translational machinery to process proinsulin―a precursor form―into fully active insulin. Proinsulin conversion involves removal of two basic pairs of amino acids―the C-peptide― and is mediated by the β cell endoproteases, PC1/3 and PC2, and the exopeptidase, carboxypeptidase-H [28] [29] . Proinsulin processing occurs within secretory vesicles once they have become acidified in the presence of high concentrations of Ca²⁺ [30] . Importantly, the glucose-induced increase in proinsulin biosynthesis is matched by a similar increase in the biosynthesis of PC1/3 and PC2 (among other proteins associated with secretory granules), which ensures proper processing of the bulk of proinsulin molecules synthesized [31] .

Combining all the unique features of the β cell, the end result is a cell with impressive sensitivity to glucose and the ability to modulate insulin secretion almost instantaneously in response to glucose. In addition, the β cell is able to maintain relatively constant stores of insulin through glucose-mediated control of insulin biosynthesis at several levels. So, beyond the ability to express insulin itself (a feat that could conceivably be achieved in any cell type using a strong constitutive promoter), which of these features are absolutely critical in a surrogate β cell and which of these features can be bypassed (see Table 1)?

Given that glucose is the primary stimulus modulating insulin secretion from β cells and the primary effector of insulin action, the impressive glucose sensitivity conferred by GLUT2 and glucokinase is an absolutely critical feature that must be present in an engineered surrogate β cell. Without these proteins, a surrogate β cell would not be able to precisely “sense” glucose concentration and control insulin output over a broad range of circulating glucose concentrations. The response of the surrogate cell would instead be unregulated. Although the expression of GLUT2 and glucokinase is somewhat restricted to β cells, other cells like hepatocytes and certain cells of the gut and hypothalamus also express both proteins. Of course, either or both of these proteins

Abbreviations: G6Pase, glucose-6-phosphatase; L-PK, L-pyruvate kinase; GLUT2, glucose transporter-2; IGFBP-1, insulin-like growth factor binding protein-1; PEPCK, phosphoenolpyruvate carboxykinase; ER, endoplasmic reticulum; PC, prohormone convertase; PTB, polypyrimidine tract-binding protein; UTR, untranslated region.

could be co-expressed with insulin to create a surrogate β cell with adequate glucose sensitivity, but this would greatly increase the complexity of genetic manipulation. Nonetheless, several groups have done so in vitro and in vivo. For instance, Callejas et al. enhanced the glucose sensing capacity of skeletal muscle through expression of glucokinase [32] . When co-expressed with insulin using an adeno-associated virus (AAV) in diabetic dogs, they were able to normalize fasting hyperglycemia and accelerate glucose disposal after oral challenge for over four years [33] . Similarly, Faradji et al. co-expressed GLUT2 and glucokinase with insulin in intermediate pituitary cells to confer glucose-sensing capabilities into these cells [34] . While their genetic manipulation conferred a degree of glucose-stimulated insulin secretion (GSIS), they more prominently observed defective glucose metabolism and severe apoptotic cell death. Thus, it is critical to not just express each component but to express them at physiological levels and take into consideration the balance of other metabolic enzymes to obtain a functional β cell [35] . Together, it would be most sensible to target a cell type that already expresses GLUT2 and glucokinase when engineering a surrogate β cell.

In addition to precise glucose sensitivity, a surrogate β cell must also be able to process proinsulin into fully active insulin. This is important because proinsulin has less than 10% of the biologic activity of fully processed insulin [36] . Processing of wild-type proinsulin requires the β cell prohormone convertases, PC1/3 and PC2 [37] . However, these processing enzymes are only found in cells with the regulated secretory pathway, such as pituitary cells and intestinal K cells. An alternative mechanism to process proinsulin without the need for PC1/3 and PC2 is to mutate the preproinsulin sequence so that it can be cleaved by ubiquitously expressed endoproteases like furin [38] [39] . In so doing, any cell of the body could process proinsulin into fully active insulin. Another strategy employed by Lee et al. to bypass the necessity for enzymatic conversion into fully active insulin is to express a single-chain insulin analogue where 35 residues of the C-peptide were replaced by a short turn-forming heptapeptide [40] . Upon delivery of this single-chain insulin analogue using a rAAV, they were able to ameliorate hyperglycemia in streptozotocin (STZ)-induced and autoimmune diabetic rats for over eight months. Thus, while it is critical for a surrogate β cell to have the capacity to process proinsulin into fully active insulin, multiple mechanisms exist to bypass the need for expression of PC1/3 and PC2. In addition, the preproinsulin cDNA sequence can be mutated to increase or decrease the stability of insulin [38] [41] , providing greater flexibility in dosing for gene therapy applications.

A surrogate β cell must also have some mechanism(s) in place to translate glucose sensitivity into altered insulin production/secretion. As mentioned above, pancreatic β cells have several mechanisms in place to alter insulin production/secretion in response to glucose. Pancreatic β cells: 1) secrete insulin almost instantaneously from storage granules through stimulus-secretion coupling, 2) upregulate insulin transcription, 3) improve preproinsulin mRNA stability, and 4) increase preproinsulin mRNA translation upon increased glucose availability. Together, this enables the β cell to rapidly secrete insulin in response to elevated blood glucose levels and maintain insulin stores at a relatively constant level on a long-term scale. So does a surrogate β cell need to have all of these mechanisms in place to control blood glucose levels, or will a subset of these mechanisms suffice?

The ability to store secreted proteins is not exclusive to β cells but can also be found in all cell types involved in regulated secretion, such as neuroendocrine cells. However, it is debatable whether it is necessary to recapitulate the regulated secretory pathway, which enables β cells to secrete insulin almost immediately upon stimulation with glucose. Keep in mind that a transient hyperglycemia alone is not life-threatening. Diabetes is such a devastating illness because of the secondary complications that arise as a consequence of sustained hyperglycemia. Thus, as long as a surrogate β cell has the ability to produce appropriate amounts of insulin within a reasonable time frame, if perhaps not immediately, in response to elevated glucose concentrations, the kinetics of insulin secretion are not that critical. The quantity of insulin output must simply be large enough to correct hyperglycemia, but not too large as to induce hypoglycemia, a potentially life-threatening consequence. Thus, any glucose-responsive mechanism capable of precisely augmenting insulin production/secretion could be a good starting point for engineering a surrogate β cell, as long as basal, unstimulated insulin output does not induce hypoglycemia under fasting conditions. To endow cells with the regulated secretory pathway, Rivera et al. engineered an insulin protein that aggregates in the endoplasmic reticulum until stimulation with a small molecule that induces protein disaggregation [42] . In so doing, they were able to generate rapid and pulsatile secretion of insulin in vivo and transiently correct diabetic hyperglycemia. However, this manipulation lacks glucose- responsiveness.

Another possible mechanism to control insulin output would be to endow a surrogate β cell with glucose- responsive control of insulin transcription. β cells achieve this through glucose-regulated changes in the intracellular localization and activity of key transcription factors, namely PDX1 and MafA. However, other cells have distinct glucose-responsive mechanisms that could be exploited to alter insulin transcription in a surrogate β cell. For instance, hepatocytes increase the production of enzymes necessary for de novo lipogenesis in response to elevated glucose concentrations [43] [44] . They do so through binding of carbohydrate response elements (ChoREs) by a glucose-regulated transcription factor, known as carbohydrate response element binding protein (ChREBP). ChoREs are composed of two 6-bp motifs with the consensus sequence CACGTG and are found in the promoters of several genes encoding lipogenic enzymes, like L-type pyruvate kinase (L-PK), S₁₄, acetyl-CoA carboxylase, and fatty acid synthase [45] . This mechanism of glucose-regulatable transcriptional control is best understood in hepatocytes but is likely also shared by cells of the small intestine and fat. Importantly, it has been utilized in several instances to control insulin output from surrogate β cells [46] - [51] , as discussed below. Alternatively, Auricchio and colleagues used the dimerizer drug rapamycin to provide pharmacological induction of insulin expression in hepatocytes [52] .

Another mechanism to control insulin output would be to incorporate sequences into the preproinsulin cDNA that lead to glucose-responsive changes in mRNA stability. Namely, it would be advantageous to incorporate sequences that lead to improved proinsulin mRNA stability when blood glucose levels are high and mRNA destabilization at low glucose concentrations. One way to do so would be to incorporate PTB binding sequences into the 3’ UTR of the cDNA construct. Conveniently, PTB is ubiquitously expressed across many (if not all) cell types and represents a common mediator of mRNA stability. An example can be found in hepatocytes, where PTB stabilizes albumin mRNA by binding an identical sequence in the 3’ UTR [53] . This is particularly important for the creation of surrogate β cells, as the half-life of preproinsulin mRNA has been reported to be less than six hours in hepatocytes [24] . The incorporation of PTB binding sites would also endow a surrogate β cell with glucose-responsive control of mRNA translation [24] . Thus, multiple levels of glucose-responsive control could be attained through this mechanism.

Overall, a surrogate β cell should, at minimum, be able to synthesize and secrete fully-processed insulin in a glucose-responsive fashion. This would require the ability to sense small changes in glucose concentration (i.e. express GLUT2 and glucokinase), respond to those changes by altering insulin output (either through altered biosynthesis or secretion), and process proinsulin into fully active insulin. Of utmost importance, constitutive insulin secretion should be limited to avoid the possibility of hypoglycemia during fasting periods, and in theory, a truly glucose-responsive system would be effective at preventing constitutive insulin release that exceeds basal needs.

The process of genetically engineering surrogate β cells begins by delivering suitably designed genes into target cells. Many gene delivery tools exist to accomplish this task and can be broadly grouped into two categories: viral and non-viral. Non-viral methods have the advantage of being less immunogenic. However, non-viral methods typically only drive transgene expression transiently, as most do not have a mechanism to induce chromosomal integration. In addition, they tend to deliver genes inefficiently in vivo. Viral methods, on the other hand, deliver transgenes with greater efficiency but will often elicit some degree of immune response. Several viruses have been used for the purpose of generating surrogate β cells, with each possessing their own unique features.

Adenoviruses were among the first viral vectors used for the generation of surrogate β cells due to their ability to transduce non-dividing cells with high efficiency. In addition, adenoviral vectors can be produced at very high titers relatively easily, enabling the type of production necessary for translating a treatment to the clinic. However, adenoviruses are unable to integrate their genetic cargo into the genome and thus provide only transient gene expression [54] [55] . Studies using adenoviral vectors to deliver the insulin gene into diabetic animals generally noted a correction of hyperglycemia for only a month [56] . Thus, repeated injection of adenoviral vectors would be necessary to sustain normoglycemia. Unfortunately, the immune response elicited by the first treatment precludes effective repeated adenovirus-based treatments. In fact, adenoviruses elicit such a profound immune response that their administration led to the death of a patient in 1999 [57] [58] . Thus, while adenoviruses are extremely efficient gene delivery tools, they are not well suited as a therapeutic tool for treatment of T1DM.

To combat issues regarding immunogenicity and short-term expression, AAVs have been used. AAVs are able to transduce both dividing and non-dividing cells and are able to integrate transgenes into the host genome at a specific site on chromosome 19, enabling long-term transgene expression [59] . The primary disadvantage of using AAVs is that their packaging capacity is less than 5 kb, limiting the use of larger expression cassettes with greater complexity for regulated expression. Furthermore, they cannot be produced in as high a titer as adenoviruses, limiting their utility as a therapeutic tool. Nonetheless, AAVs have been used successfully to generate surrogate β cells. Park et al. used a rAAV to deliver furin-cleavable insulin under control of the CMV promoter into STZ-induced diabetic rats and found improved glucose tolerance (at 2 g/kg) comparable to that of non-dia- betic control rats [60] . Additionally, they observed less inflammation and fewer side effects using rAAV when compared to the same treatment using adenoviral vectors.

Retroviral vectors are perhaps the most widely used gene delivery vector, owing to their natural ability to integrate their genetic cargo into a host’s genome and obtain sustained gene expression. However, retroviral integration has been shown to have some preference to occur in close proximity to the transcriptional regulatory sequences of proto-oncogenes, as observed in 1999 following the treatment of nine severe-combined immunodeficiency patients [61] . Insertional mutagenesis led to the development of leukemia in four of the nine patients, and as cautionary measure, the field of clinical gene therapy was temporarily halted. Retroviral vectors are also limited by their inability to transduce non-dividing cells, a problem that severely hinders their utility for the production of surrogate β cells from quiescent cells. Accordingly, retroviral vectors have only been used sparingly in insulin gene therapy applications, and in these cases, hepatocyte proliferation must first be stimulated before retrovirus infusion. For instance, Muzzin and colleagues performed a partial hepatectomy before using retroviral vectors to deliver furin-cleavable preproinsulin-1 into STZ-induced diabetic rats [62] . Remarkably, they were able to attain near normoglycemia, but the necessity for hepatocyte proliferation precludes the translation of retroviral vectors in humans.

Lentiviral vectors provide a particularly attractive gene delivery vehicle to genetically engineer surrogate β cells, owing to their ability to transduce dividing and non-dividing cells and integrate their cargo into the host’s genome. Lentiviral vectors are a type of retrovirus and thus share some of the same concerns of insertional mutagenesis. However, lentiviral vectors display a more random integration profile with less preference to integrate near regulatory sequences of proto-oncogenes than retroviral vectors, reducing the risk of insertional mutagenesis [63] [64] . Additionally, lentiviral vectors do not elicit a strong immune response. Unfortunately, it remains a challenge to produce lentiviral vectors in high enough titer for clinical application [65] . Nonetheless, lentiviral vectors offer stable, long-term transgene expression with an improved biosafety profile and are thus an excellent candidate as a therapeutic tool for treatment of T1DM. Ren and colleagues used lentiviral vectors to deliver furin-cleavable insulin to the livers of STZ-induced diabetic rats [66] and non-obese diabetic mice [67] . In each case, correction of diabetic hyperglycemia was observed for at least five months post-treatment with no evidence of impaired liver function, intrahepatic inflammation, or recurring autoimmunity against the newly-formed insulin-producing cells.

Given the prevalence and gravity of diabetes, significant research efforts have been put forth to treat the disease. Some of these efforts have been aimed at preventing the autoimmune destruction of native β cells [68] - [71] , whereas a more significant portion of research has been aimed at generating surrogate β cells, either through expansion of remaining β cells in vitro [72] , differentiation of stem cells into β cells [73] - [75] , or genetic modification. Indeed, attempts to differentiate embryonic stem cells (ESCs) into β cells have been quite successful but are outside the scope of this review [76] . Here, we will review the different strategies used to genetically engineer surrogate β cells, beginning with the overexpression of β cell-specific transcription factors and concluding with insulin gene therapy.

All current therapies for T1DM possess significant drawbacks. In addition to effectively correcting hyperglycemia through precise glucose-responsiveness, a treatment for T1DM should be safe, affordable, readily available, and long-lasting without the need for lifelong immunosuppression. The genetic engineering of surrogate β cells provides an exciting alternative to treat T1DM with the potential to meet all of these needs. For instance, by delivering a glucose-responsive insulin expression cassette into a cell type with GLUT2 and glucokinase expression using an integrating viral vector, long-term correction of diabetic hyperglycemia could be attained. However, if such a therapy is to have success in the clinic, several challenges must be addressed.

Lentiviral vectors provide a promising delivery tool for the production of surrogate β cells but are severely hampered by our inability to produce them in high enough titer to affordably translate this technology to the clinic. In addition, while lentiviral vectors have the capacity to transduce non-dividing cells, transduction efficiency has been shown to be greater in cells that are actively dividing [115] . Thus, improvements to lentiviral vector design that enhance transduction efficiency and/or transgene expression would reduce the necessity for large viral doses. This is particularly important for insulin gene therapy because the insulin molecule has a relatively short circulating half-life, estimated at around 4 - 6 minutes [116] . Adenoviral vectors, on the other hand, are able to be produced in extremely high titers and are very efficient at transducing non-dividing cells but are only capable of producing transient transgene expression. As a result, they do not provide a viable option for the production of surrogate β cells. However, recent advances have allowed researchers to endow adenoviruses with the ability to integrate their cargo by incorporating the Sleeping Beauty transposase system into the expression cassette [117] . To overcome or minimize immunity to adenoviral vectors, a newer version of “gutted” adenovirus with minimal wild-type adenovirus sequences is currently being used in a variety of human clinical trials.

An exciting option to generate surrogate β cells that closely mimic native β cells is through forced expression of β cell-specific transcription factors. However, this strategy is greatly limited by concerns of recurring autoimmunity, as the newly-formed β cells will inevitably express a more expansive repertoire of β cell proteins, including autoantigens. To protect against autoimmunity, successful execution of this strategy will ultimately require immunosuppression, especially if the newly-formed β cells are generated ex vivo. An alternative strategy to protect ex vivo-generated β cells from autoimmune destruction would be to implant them in an immunoprotective implantation device. However, no biomaterial is completely inert, and proteins from serum and extracellular matrix will accumulate on the implantation device over time, which will affect diffusion of nutrients and waste products into and out of the device. More importantly, this will affect the precision of glucose sensitivity and insulin secretion over time, as well.

Lastly, it will be essential to get sufficient glucose-induced insulin expression from surrogate β cells to precisely control glucose metabolism. For insulin gene therapy, this requires glucose-regulatable expression of insulin in a cell type that is capable of this task. We have achieved this by restricting insulin expression to hepatocytes by using the albumin promoter and incorporating GIREs from the S₁₄ gene to amplify insulin expression in response to elevated glucose levels. Using the relatively weak albumin promoter has the added advantage of reducing basal insulin expression, thus reducing the risk of hypoglycemic episodes. Similarly, other groups have devised glucose-regulatable strategies to produce surrogate β cells of clinical relevance. Ultimately, the success of such a strategy will hinge on the amplitude of glucose induction and ability to reproducibly generate a therapeutic quantity of surrogate β cells. As the current gene delivery tools continue to advance, these goals will become more attainable for treatment of T1DM.

Andrew M. Handorf,Hans W. Sollinger,Tausif Alam, (2015) Genetic Engineering of Surrogate β Cells for Treatment of Type 1 Diabetes Mellitus. Journal of Diabetes Mellitus,05,295-312. doi: 10.4236/jdm.2015.54037