Type 1 diabetes mellitus is an autoimmune disease resulting from the destruction of pancreatic β cells. Current treatments for patients with type 1 diabetes mellitus include daily insulin injections or whole-pancreas transplant, each of which are associated with profound drawbacks. Insulin gene therapy, which has shown great efficacy in correcting hyperglycemia in animal models, holds great promise as an alternative strategy to treat type 1 diabetes mellitus in humans. Insulin gene therapy refers to the targeted expression of insulin in non-β cells, with hepatocytes emerging as the primary therapeutic target. In this review, we present an overview of the current state of insulin gene therapy to treat type 1 diabetes mellitus, including the need for an alternative therapy, important features dictating the success of the therapy, and current obstacles preventing the translation of this treatment option to a clinical setting. In so doing, we hope to shed light on insulin gene therapy as a viable option to treat type 1 diabetes mellitus.

Key words : β cell, Liver, Autoimmunity, Pancreas transplant, Minicircle

Introduction

Type 1 diabetes mellitus (T1DM) results from the autoimmune destruction of insulin-producing β cells of the pancreas. As a result, affected individuals are unable to produce adequate insulin to precisely regulate blood glucose levels. Over time, chronic hyperglycemia results in numerous complications affecting the vascular system, kidneys, retina, lenses, nervous system, and skin. In fact, diabetes is the leading cause of end-stage kidney failure, blindness, nontraumatic amputations, and a variety of debilitating neuropathies. Despite active research, there is currently no cure for T1DM.

According to the National Diabetes Statistics Report, 29.1 million people, or 9.3% of the US population, have diabetes, with T1DM accounting for roughly 5% of all diagnosed cases in the adult population. The total economic cost of diabetes is estimated to be $245 billion per year, with T1DM costing an estimated $8 billion to $14 billion per year. Thus, diabetes poses a significant burden to our economy. While there is no cure for T1DM, several therapies currently exist to control blood glucose levels and limit adverse systemic damage resulting from chronic hyperglycemia. Of these treatments, exogenous insulin therapy and whole-pancreas transplant are most common but present significant drawbacks. Hence, there is clearly a need for alternative state-of-the-art therapies. To that end, insulin gene therapy has arisen as a promising alternative with considerable potential for the long-term treatment of T1DM.

In this review, we will summarize the current therapies used to treat T1DM and discuss the inherent limitations of each treatment. We will then introduce a novel treatment for T1DM—insulin gene therapy—and review the important features required for its success, as well as the current state of this promising treatment. Lastly, we will discuss the current obstacles preventing insulin gene therapy from being a clinical reality and propose future directions critical for the translation of this therapy. In doing so, we hope to shed light on insulin gene therapy as a promising alternative to treat T1DM.

Traditional therapies for treatment of Type 1 diabetes mellitus
Exogenous insulin-based therapies
Type 1 diabetes mellitus is a chronic disease for which there is no cure. Instead, classical treatments have been aimed at obtaining an approximate restoration of deficient insulin levels. The most common form of insulin therapy involves injection of exogenous insulin, which typically requires multiple insulin shots per day. While daily insulin injections preserve life and delay the onset of secondary complications like blindness or kidney failure, they are cumbersome and inadequate at restoring optimal glucose control. In addition, exogenous insulin therapy is associated with adverse events like hypoglycemia, weight gain, and worsening diabetic retinopathy if HbA1c levels decrease too rapidly.1 The insufficiencies of exogenous insulin treatment arise from the fact that most insulin regimens are nonphysiologic in nature and unable to mimic normal β cell secretion in response to ever-changing blood glucose levels. To improve upon traditional insulin therapy, more physiologically mimetic insulin replacement therapies that replace prandial and basal insulin separately,2 insulin analogues with altered stability or activity,1 and insulin pumps3 have been used. Nonetheless, the sophistication of insulin secretion from β cells is impossible to duplicate with exogenous insulin therapy. Consequently, exogenous insulin therapy delays, but does not prevent, the onset of complications that ultimately increase morbidity and mortality.

Transplant therapies
To replace endogenous β cell function and obtain tighter control of blood glucose levels, whole-pancreas or pancreatic islet transplant surgeries have been performed. A whole-pancreas transplant was first performed in 1966,⁴ and today, over 30 000 pancreatic transplants have been completed.⁵ Pancreas transplants have demonstrated sustained insulin independence and excellent control of blood glucose for several recipients posttransplant, as evidenced by normal HbA1c, a measure of average plasma glucose over the previous 2 to 3 months. However, the usefulness of pancreas transplant therapy is limited by a severe shortage of donor organs and the need for lifelong immuno­suppression, along with its associated adverse effects.⁶ Thus, this procedure is only recommended for T1DM patients who are unresponsive to conventional insulin-based therapies.⁷ Transplant of pancreatic islets was proposed as a better alternative to whole-pancreas transplant on the basis of 2 theoretical advantages: the procedure is less invasive and a single donor’s islets could serve multiple patients if the cells could be induced to proliferate ex vivo or in vivo. Islet transplant therapy has enabled patients to remain insulin independent for an average of approximately 1 year, but long-term graft survival rates have remained < 10%.^8,9 Despite considerable efforts, equivalent successes like those observed with whole-pancreas transplants have not been matched by islet transplants.⁸

Gene therapy for treatment of type 1 diabetes mellitus
Due to the inherent limitations of exogenous insulin- and transplant-based therapies, alternative strategies are being investigated to treat T1DM. The goal of future treatments will be to restore dynamic control over blood glucose levels without requiring cumbersome daily injections, major surgical operations, or lifelong immunosuppressive regimens. Additionally, future therapies must be affordable for all patients. Gene therapy has emerged as a promising alternative for the treatment T1DM that could meet all of the aforementioned criteria. Gene therapy can be broadly defined as the treatment of a disease by using therapeutic genes as drugs. These therapeutic genes are delivered to target cells using a vector, such as a virus, to restore or replace lost cellular functions. In the case of T1DM, the delivery of therapeutic genes can improve the clinical outcome of diabetic individuals by preventing the autoimmune des­truction of β cells prior to the onset of disease, reprogramming non-β cells into surrogate β cells, or simply replacing the function of lost β cells. Here, we will briefly describe the first 2 gene therapy strategies and their limitations before discussing the last option—replacing the function of β cells via insulin gene therapy—in-depth. Specifically, we will cover the important features required for successful insulin gene therapy, the current obstacles preventing the therapy from reaching clinics, and important future directions.

Prevention of autoimmune β cell destruction
The idea of preventing the autoimmune destruction of β cells through the use of gene therapy is a logical one. To do so, researchers have attempted to (1) alter the immune system so that it no longer recognizes β cell antigens as foreign or (2) modify the residual β cells so that they can withstand attack from the patient’s own immune system. For example, researchers have overexpressed IL-4 via an adenoviral vector in nonobese diabetic mice,10 or overexpressed the antiapoptotic gene, bcl-2, in β cells specifically,11 to attenuate the autoimmune attack and increase the survival of β cells. However, these approaches are severely limited for 3 reasons. First, this strategy relies on the early detection of diabetes, and often times, greater than 80% of an individual’s β cells have already been destroyed by the time they become symptomatic. Thus, efforts to protect the remaining β cells may prove futile. Second, T1DM is a multifactorial disease, making it nearly impossible to predict whether a prediabetic individual will ever succumb to the disease.12 Thus, early intervention can be risky and perhaps even accelerate the progression of the disease. Third, the immune system is highly evolved and its complexities are not well understood; however, what is becoming increasingly apparent are the innumerable functional re­dundancies that allow it to compensate for the loss of any single factor or pathway. At our current level of understanding, it seems inconceivable to selectively target the immune system to prevent the autoimmune destruction of β cells; thus, other gene therapy strategies have been explored.

Reprogramming non-β cells into β cells
The goal of reprogramming non-β cells into surrogate β cells is to create replacement cells that are as similar as possible to native β cells. Researchers have targeted several cell types with the hopes of reprogramming them into β cells, including pancreatic exocrine cells,^13,14 keratinocytes,¹⁵ hepatic oval stem cells,¹⁶ adipose-derived stem cells,¹⁷ and hepatocytes. Of these, hepatocytes have been most commonly targeted due to the fact that they are closely related developmentally to β cells.

To accomplish this reprogramming, the transcription factor pancreatic and duodenal homeobox gene 1 (PDX1), which regulates pancreatic development during embryogenesis and controls β cell function in adults, has proven indispensable for the conversion of non-β cells into β cells. Ectopic expression of PDX1 has proven quite successful at converting non-β cells into β cells capable of synthesizing, processing, and secreting insulin.^18-20 In conjunction with PDX1, other transcription factors like NKX6.1,²¹ Neurogenin^3,13,22 and NeuroD²² have been shown to enhance reprogramming efficiency. Importantly, many studies have found that surrogate β cells produced from reprogramming are able to ameliorate streptozotocin-induced hyperglycemia in mice.¹⁸ Despite these successes, the overall efficacy of reprogramming strategies relies on the long-term absence of recurring autoimmunity against newly formed β cells, which inevitably express a variety of autoantigens that ultimately led to the destruction of native β cells to begin with.²³ While studies in nonobese diabetic mice, a model of autoimmune diabetes, provide hope that autoimmunity could be averted through reprogramming strategies,²⁰ these studies must be performed for longer time periods to assess true efficacy. Ultimately, these strategies will require either lifelong immunosuppression or selective immunomodulation to prolong the survival of the newly generated β cells.

Insulin gene therapy
Given the autoimmune etiology of T1DM, it would be paradoxically advantageous to treat the disorder without regenerating β cells, as the risk of their autoimmune destruction is theoretically great. Instead, it may be advantageous to take a minimalist approach and simply replace the key functions of β cells without substantially altering the phenotype of the host cell. Along these lines, researchers have actively investigated the possibility of expressing insulin alone in non-β cells, a field known as insulin gene therapy.

For insulin gene therapy strategies to be successful, several criteria must be considered. First, an appropriate target organ must be selected. Ideally, the target organ would have the ability to sense and respond to continually changing blood glucose levels. In addition, it would be advantageous for the target organ to have the capacity to store insulin and secrete it in a rapid and glucose-inducible fashion. Second, an effective gene delivery method must be used that is both safe and effective at driving long-term insulin expression in the target organ. Lastly, ectopic insulin expression should be responsive to fluctuating blood glucose levels, being up-regulated during hyperglycemia and down­regulated during euglycemia. The following sections will outline important considerations to optimize each of these criteria and summarize the current state of the field.

The most important feature that a target cell for insulin gene therapy should possess is the ability to respond to fluctuating levels of glucose. This implies that the cells express both glucose transporter-2 (GLUT2) and glucokinase (GK), enabling transport of glucose into a cell and its subsequent metabol­ism.²⁴ The only cells that express both GLUT2 and GK are pancreatic β cells, hepatocytes, and cells of the hypothalamus and small intestine. Of these, hepatocytes are particularly good candidates for insulin gene therapy because they are essential regulators of glucose metabolism in response to insulin. Of course, GLUT2 and GK can be co-expressed with insulin in other cell types, but this greatly increases the complexity of gene therapy. For example, Bosch and colleagues targeted skeletal muscle cells, one of the primary targets of insulin action, and found that GK needed to be co-expressed with insulin to attain normoglycemia.²⁵ Similarly, Hughes and coworkers found that AtT-20ins cells—anterior pituitary cells that express GK but not GLUT2—needed to be cotransfected with insulin and GLUT2 to confer glucose-stimulated insulin secretion.^26,27

It would also be beneficial for the cellular target of gene therapy to be immunoprivileged, thus allowing it to evade preexisting autoimmunity. This is particularly critical, given that insulin has been shown to be one of the primary autoantigens targeted by the adaptive immune system.²³ Importantly, previously published studies have shown that insulin misexpression in the liver does not cause hepatocytes to become the target of autoimmunity.²⁸

Lastly, it would be ideal if the target cells could process proinsulin—a precursor form of insulin with greatly reduced biological activity—into mature insulin and store it in granules for immediate secretion upon hyperglycemia. For proper processing of proinsulin, the β cell prohormone convertases PC2 and PC3 are required.²⁹ Unfortunately, these enzymes are only expressed in neuroendocrine secretory cells. To complicate matters further, few cells in the body have the capacity to store insulin (or any other protein) in secretory granules. Thus, groups have devised novel strategies to bypass these concerns. Irminger and associates expressed PC2 and PC3 in rat insulinoma cells and found that proinsulin could then be processed into mature insulin.³⁰ Using this strategy, virtually any cell of the body could be induced to express fully processed insulin. However, misexpressing exogenous proteases in vivo can have unforeseen consequences that greatly hinder cellular activities, perhaps limiting the use of this strategy. Thus, other strategies have been used to bypass this concern.

To compensate for most cells’ inability to store insulin, Rivera and associates engineered an insulin analogue that aggregated in the endoplasmic reticulum and was only secreted upon stimulation with a synthetic small molecule drug that induces protein disaggregation, in essence turning the endoplasmic reticulum into a proinsulin storage depot. Indeed, they showed that this method led to rapid secretion of insulin and lowered diabetic hyperglycemia in mice.³¹ Similarly, Auricchio and associates obtained regulated secretion of insulin using a rapamycin-inducible system in vivo.³² However, when using drug-inducible systems, researchers must select the drug of choice carefully to limit unwanted adverse effects. For instance, rapamycin produces diabetes-like symptoms, such as insensitivity to insulin and decreased glucose tolerance, and as such, would not be desirable for insulin gene therapy applications.

To deliver insulin to the target cell, researchers have used several gene delivery vehicles. The ideal gene expression vector for insulin gene therapy should have the ability to target specific cells, transduce both dividing and nondividing cells, be nonimmunogenic, have reliable methods for large scale production, and most importantly, induce long-term expression of insulin. Both viral and nonviral vector-based gene delivery methods have been used for insulin gene therapy applications, with each showing successful amelioration of diabetes-associated hyperglycemia in small animal models.^33-35 However, nonviral gene delivery methods are limited by their inefficient delivery to target cells and lack of chromosomal integration, which restricts the longevity of gene expression. As such, viral vectors have been used more prevalently in insulin gene therapy applications and are ultimately the future of the therapy. Adenoviruses,^34,36,37 adeno-associated viruses,^25,38,39 oncoretroviruses,^40,41 and lentiviruses^42-44 all have been used to deliver insulin to hepatocytes, but they vary in their ability to meet the essential criteria described previously. A summary of each viral vector’s ability to meet the demands required for insulin gene therapy applications is included in Table 1. Based on the fact that lentiviral vectors can transduce both dividing and nondividing cells, are nonimmunogenic, and can induce long-term expression of insulin, they are considered the optimal gene delivery vehicle for long-term correction of T1DM and its associated complications, and indeed, they have proven successful at delivering insulin to the liver in several different studies.^42-44

The most critical aspect dictating the success of insulin gene therapy strategies is the sophistication of the DNA construct. The ideal DNA construct must be able to produce a sufficient quantity of biologically active insulin in a glucose-responsive fashion. In addition, it would be beneficial if the DNA construct design restricted the expression of insulin specifically to the target cell and included additional components to precisely attenuate the amount of insulin produced as hyperglycemia subsides. A construct possessing these features would hold great utility in insulin gene therapy.

Given that most cells, including hepatocytes, do not express PC2 or PC3, the first feature that an insulin construct should possess is a proinsulin cDNA sequence capable of being processed by the target cells into mature insulin. As mentioned previously, wild-type proinsulin expressed in cells other than β cells will lack potent bioactivity due to the absence of the proinsulin processing machinery. The most commonly used strategy to overcome this problem has been to incorporate furin cleavage sequences within the preproinsulin cDNA sequence. In so doing, the modified proinsulin can be cleaved by furin—a ubiquitously expressed endoprotease. Simonson and colleagues incorporated furin cleavage sites between the B-chain and C-peptide, and between the C-peptide and A-chain of proinsulin, and found a significant increase in the secretion of fully processed insulin from rat myoblasts, which ultimately resulted in improved glucose oxidation.⁴⁵ This provides the opportunity for virtually any cell type in the body to produce fully mature insulin, albeit in a constitutive and unregulated fashion.

The next feature of an ideal DNA construct would be an element that restricts expression to the targeted cell type of choice. To do so, many groups have used tissue-specific promoters, and for hepatic insulin gene therapy, a variety of liver-specific promoters have been used. Examples of liver-specific promoters include the insulin-like growth factor binding protein-1 (IGFBP-1),⁴⁶ glucose-6-phosphatase (G6Pase), liver-type pyruvate kinase (L-PK),⁴⁷ phosphoenolpyruvate carboxykinase (PEPCK),²⁸ albumin,^34,37 and S-14. While liver-specific trans­criptional regulation of insulin gene transcription has been achieved using these promoters, significant limitations have been observed. These gene expression systems displayed relatively weak promoter activity, especially when compared with strong constitutive promoters like cytomegalovirus. As a result, the amount of secreted insulin was often insufficient to fully correct hyperglycemia. For instance, Han and colleagues used the L-PK basal promoter to obtain glucose-responsive, liver-specific insulin expression and found that it displayed dramatically lower luciferase activity than the constitutive cytomegalovirus promoter. Even after adding a series of enhancer elements upstream of the promoter, its transcriptional activity remained weaker than that of the cytomegalovirus promoter. None­theless, the modified L-PK promoter showed glucose-responsiveness and was able to restore normoglycemia for up to 1 month, although glucose clearance was delayed and slower than normal.⁴⁷ It should be noted that 1 potential limitation of using the L-PK promoter is that it is inhibited by insulin, thus potentially creating an undesirable negative feedback on insulin expression. To avoid this scenario while still attaining liver-specific transgene expression, albumin and S14 promoters could be used.

To enhance insulin expression from weak promoters, enhancer elements are commonly incorporated into the DNA construct. A useful enhancer for insulin gene therapy is the glucose-inducible responsive element. Thule and associates constructed a glucose-responsive IGFBP-1 promoter to control insulin expression in the liver by inserting glucose-responsive elements from the rat L-PK gene. In so doing, they were able to induce insulin expression in a glucose-dependent manner from cultured primary rat hepatocytes in vitro and correct streptozotocin-induced hyperglycemia in rats in vivo.^46,48 However, the IGFBP-1 promoter, like the L-PK promoter, is inhibited by insulin. Conversely, our work has used an insulin expression plasmid containing 3 copies of the S14-based glucose-inducible responsive elements to confer glucose dependence to the liver-specific rat albumin pro­moter.

Using this system to control the expression of furin-cleavable insulin, we demonstrated that the glucose-inducible production of processed insulin was able to reduce fasting blood glucose levels, blood glucose levels of rats fed ad libitum, and peak blood glucose levels during oral glucose tolerance tests in streptozotocin-treated diabetic rats. While sig­nificantly improved, blood glucose levels of rats fed ad libitum and during the oral glucose test failed to reach that of nondiabetic rats.³⁷

To improve upon the efficiency of this initial glucose-responsive insulin construct, we incorpo­rated additional elements known to enhance gene transcription, mRNA processing, and translational efficiency. Specifically, we tested the ability of a transcriptional enhancer from the human α-fetoprotein gene, an intron from the human growth hormone sequence that enhances RNA processing efficiency, a translation enhancer from the human vascular endothelial growth factor sequence that functions as an internal ribosomal entry site, and an intron from the 3’-untranslated region of the human albumin sequence that facilitates mRNA processing to enhance insulin production and better restore normoglycemia in streptozotocin-treated diabetic rats. In so doing, we found that incorporation of the vascular endothelial growth factor-derived trans­lational enhancer alone resulted in a 4- to 6-fold increase in insulin production in both low and high glucose conditions. Furthermore, we found that individual incorporation of the human growth hormone intron or α-fetoprotein transcriptional enhancer enhanced insulin production. However, when both elements were incorporated together, insulin production was not further improved. Thus, we selected a DNA construct that contained the α-fetoprotein transcriptional enhancer, vascular endothelial growth factor translational enhancer, and albumin 3’- untranslated region in addition to the 3 copies of glucose-inducible responsive elements, albumin promoter and human insulin gene (Figure 1).³⁴

When testing this insulin construct for its ability to improve the overall phenotype of streptozotocin-treated diabetic rats, we were not only able to induce normoglycemia in fasting rats but also rats fed ad libitum (Figure 2). In treated diabetic rats, we were also able to correct weight loss due to uncontrolled hyperglycemia such that the rate of weight gain matched that of healthy control rats for at least 1 month (Figure 3). In addition, intraperitoneal glucose tolerance tests demonstrated a correction of hyperglycemia within 45 minutes. Importantly, a single treatment with our DNA construct significantly improved many systemic abnormalities downstream of hyperglycemia. Specifically, it raised serum albumin levels in diabetic rats to normal and restored elevated serum levels of aspartate transaminase, alanine aminotransferase, and alkaline phosphatase to near normal. The treatment also significantly reduced hyperglycemia-associated hypertriglyceridemia and hypercholesterolemia, indicating healthy liver function. Interestingly, even after precise glycemic control was lost (~1 month), the metabolic benefits of treatment with our DNA construct persisted well beyond that, suggesting that even suboptimal insulin production can counteract much of the immediate systemic damage associated with hyperglycemia. Based upon these results, our liver-based insulin gene therapy treatment offers a promising approach to treat T1DM.³⁴

Future perspectives
Many advances in hepatic insulin gene therapy have been made over the past 2 decades, fueling optimism that a treatment for T1DM may be found sooner rather than later. However, several obstacles still remain before this approach can become a clinical reality. First, it is important to optimize the hepatic delivery of vectors to drive glucose-responsive insulin production. Currently, physiologic control of blood glucose levels has been obtained using glucose-responsive, liver-specific promoters delivered via adenoviral vectors and minicircles, as well as via lentiviral vectors with strong constitutive promoters. Ultimately, it will be necessary to combine the strengths of lentiviral vectors and glucose-responsive, liver-specific promoters to obtain long-term, dynamic correction of diabetes without the threat of hypoglycemic episodes. In fact, such a treatment could potentially provide lifelong correction of T1DM. To do so, it may be necessary to add additional elements to our current glucose-responsive insulin constructs to further enhance expression of insulin.

Second, although insulin gene therapy has yielded promising results in mouse and rat models of T1DM, these models do not always accurately replicate human disease, especially when the immune system plays a role in the pathology of the disease. Future studies, like those conducted by Bosch and colleagues, must be conducted in large animal models, such as dogs, pigs, or nonhuman primates, to provide a better indication of the efficacy of insulin gene therapy before contemplating human clinical trials.

Lastly, it will be critical to assess the long-term safety of lentiviral vector-mediated insulin gene therapy. At this time, malignant transformation associated with vector-mediated insertional muta­genesis has only been observed in 3 clinical entities, all of which occurred using first generation gamma­retroviral vectors. However, with the advent of later-generation lentiviral vectors, no adverse events have been observed, and there are currently over 2000 ongoing human gene therapy clinical trials worldwide. In addition, there are several safety mechanisms, such as self-inactivating vectors, insulators, and suicide genes, which have been built into viral vectors for gene therapy applications, thereby providing many preemptive options to improve the safety of vectors. Overall, the future appears very bright for insulin gene therapy as an alternative strategy to treat T1DM.

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