**1. Introduction**

As per the American Diabetes Association, 2-3 million individuals in the United States have type 1 diabetes (www.medicinenet.com/script/main/art.asp?articlekey=159355), with 1 in every 400 to 600 children and adolescents affected by the disease in 2007. Furthermore, similar to other autoimmune diseases, allergies and asthma, the incidence of type 1 diabetes is on the increase at an alarming rate in industrialized countries for unknown reasons. Information released by the American Diabetes Association shows 23% and 21% increased rates of type 1 and 2 diabetes, respectively, from 2001 to 2009. The increase in incidence of type 1 diabetes is especially apparent in young children and has generated an urgent need for novel treatments that can safely control diabetes-causing inflammation, and alleviate the need for administra‐ tion of exogenous insulin. Indeed, type 1 diabetes has been treated for almost a century in the same fundamental manner using daily insulin injections. Although it is a life-saving treatment and one of the most remarkable accomplishments of medicine, administration of exogenous insulin is still not a cure because it does not address the underlying autoimmunity that targets insulin-secreting beta cells. Because it is currently impossible to mimic regulation of physio‐ logical insulin levels faithfully, many type 1 diabetic individuals receiving standard of care are exposed to acute and chronic complications that cause increased morbidity and mortality. As a result, there have been intense efforts to develop immunotherapies that can eliminate or at least alleviate the need for exogenous insulin. In this case the goal is to arrest pathological autoimmunity that destroys beta cells so that the cells can regain function, and possibly proliferate and regenerate.

Two major immunotherapeutic paths have been taken. The first path relies on different forms of systemic suppression of inflammation that inhibit effector T lymphocytes in a non-specific manner. Serious side effects associated with the use of systemic immunotherapies are in‐ creased risks of cancer and infection resulting from the decreased activity of effector cells involved in beneficial destructive immune responses against cancer cells and pathogens. These

© 2013 Li and Escher; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

side effects have been observed with broadly acting immunosuppressants used to prevent organ transplant rejection, which have been also investigated for treating type 1 diabetes [1, 2]. Serious side effects can also be seen, albeit to a lesser degree, with more specific agents like antibodies that target specific molecules involved in inflammation [3, 4]. In contrast, the second immunotherapeutic path is based on administration of a self-antigen synthesized by pancreatic beta cells that is a target of pathological autoimmunity, i.e., an autoantigen. This path is thought to be safer because it aims to induce a regulatory immune response that targets the inflamed islets. Here the goal is to manipulate endogenous immune mechanisms of homeostasis that can re-establish some form of tolerance to the chosen autoantigen, as well as to other neigh‐ boring beta cell autoantigens through a mechanism known as "by-stander suppression" [5]. Accordingly, it is anticipated that pathological autoimmunity and inflammation of islets can be stopped in an organ-specific manner that does not impair the immune system.

a DNA vaccine for treatment of melanoma delivered with gold particles into skin was found to be as efficacious as 1000 microgram injected intramuscularly [12]. These results illustrate the significant impact that choice of route and method of delivery of a DNA vaccine can have not only on efficacy, but also on cost of treatment. Other delivery routes like intravenous, nasal, and sublingual have also been investigated [13]. Post delivery, expression of coding sequences in plasmid DNA results in significant levels of protein production that may persist for six

DNA Immunotherapies for Type 1 Diabetes http://dx.doi.org/10.5772/55727 535

**Figure 1. Plasmid DNA Immunotherapy for Type 1 Diabetes.** Plasmid DNA immunotherapy for type 1 diabetes can be divided into two categories: DNA vaccines and gene therapies. Plasmid DNA can be delivered using different routes, for example, the intradermal and intramuscular routes. The therapies can induce tolerogenic dendritic cells (DC), T regulatory cells (Treg), and death/anergy of T effector cells (Teff) which are the main cause of pancreatic islet

DNA vaccination was originally investigated to induce immunogenic responses to pathogens and cancer cells, but it has increasingly been applied to the induction of immune tolerance for treatment of autoimmune diseases like type 1 diabetes. DNA vaccines and other gene-based vaccines belong to a third generation of vaccines after live and attenuated whole organism vaccine and recombinant protein vaccines. These vaccines can be used to either prevent (prophylactic vaccine) or treat (therapeutic vaccine) disease depending on their potency, in which prevention is generally easier to achieve than treatment. Recent reports of beneficial results in different clinical trials using delivery of autoantigens indicate that DNA vaccination is reaching a stage where we are likely to see accelerated development of a therapeutic future for vaccines targeting a variety of autoimmune diseases. In the case of type 1 diabetes, early results using a DNA vaccine encoding insulin have shown promise in humans. In addition,

destruction.

weeks and longer without serious side effects in human patients [14, 15].

In this chapter, we will review how plasmid deoxyribonucleic acid (DNA) has been used as an immunotherapeutic vector platform to treat type 1 diabetes through each immunothera‐ peutic path. For the purpose of this review, we have called the first path "gene-based immu‐ notherapy", meaning that plasmid DNA does not encode a known autoantigen, and the second path "DNA vaccine immunotherapy" meaning that a beta cell autoantigen is encoded by plasmid DNA (Figure 1). In other words, gene-based immunotherapy relies on the inherent function of a product encoded by plasmid DNA that can in turn affect cell function. In contrast, DNA vaccine immunotherapy relies on the tolerogenic immune response induced directly by the autoantigen after its processing by immune cells. As we shall see, some DNA vaccine immunotherapies have also a gene-based immunotherapy component that acts as a molecular adjuvant to promote tolerogenic immune responses. Nevertheless, in all cases synthesis of the molecule encoded by plasmid DNA, which is almost always a protein, starts after delivery of the plasmid DNA and its uptake by cells.

Plasmid DNA has several notable advantages compared to other vectors and therapeutic molecules. For example, it consists of relatively low molecular weight circles of double stranded DNA that can be readily isolated from bacteria in a generic and cost-effective manner. In addition, plasmid DNA permits rapid turnaround when developing new candidate products, refrigeration-free storage, and synthesis over time of a chosen antigen in its native conformation. Furthermore, plasmid DNA can be given in repeat doses within short periods of time without inducing an immune response to vector and other side effects. This is in contrast with viral-based vaccines that can induce immune responses to vector and protein/ peptides that can cause anaphylaxis [6, 7].

Plasmid DNA can be delivered either as "naked" DNA or packaged into molecular scaffolds like liposomes, cationic lipids, virosomes, and polymer-based nano- and micro-particles to increase efficacy of delivery to cells [8]. Although they may sometime have pro-inflammatory properties that can be detrimental to controlling inflammation, these particles have been used to induce immune tolerance with plasmid DNA. For example, chitosan-DNA nanoparticles encoding an ovalbumin antigen are tolerogenic when delivered orally [9].

Indeed, route of delivery can play a significant role in the type and strength of immune responses induced by DNA vaccines in animal models [10, 11]. In humans, two microgram of a DNA vaccine for treatment of melanoma delivered with gold particles into skin was found to be as efficacious as 1000 microgram injected intramuscularly [12]. These results illustrate the significant impact that choice of route and method of delivery of a DNA vaccine can have not only on efficacy, but also on cost of treatment. Other delivery routes like intravenous, nasal, and sublingual have also been investigated [13]. Post delivery, expression of coding sequences in plasmid DNA results in significant levels of protein production that may persist for six weeks and longer without serious side effects in human patients [14, 15].

side effects have been observed with broadly acting immunosuppressants used to prevent organ transplant rejection, which have been also investigated for treating type 1 diabetes [1, 2]. Serious side effects can also be seen, albeit to a lesser degree, with more specific agents like antibodies that target specific molecules involved in inflammation [3, 4]. In contrast, the second immunotherapeutic path is based on administration of a self-antigen synthesized by pancreatic beta cells that is a target of pathological autoimmunity, i.e., an autoantigen. This path is thought to be safer because it aims to induce a regulatory immune response that targets the inflamed islets. Here the goal is to manipulate endogenous immune mechanisms of homeostasis that can re-establish some form of tolerance to the chosen autoantigen, as well as to other neigh‐ boring beta cell autoantigens through a mechanism known as "by-stander suppression" [5]. Accordingly, it is anticipated that pathological autoimmunity and inflammation of islets can

be stopped in an organ-specific manner that does not impair the immune system.

the plasmid DNA and its uptake by cells.

534 Type 1 Diabetes

peptides that can cause anaphylaxis [6, 7].

In this chapter, we will review how plasmid deoxyribonucleic acid (DNA) has been used as an immunotherapeutic vector platform to treat type 1 diabetes through each immunothera‐ peutic path. For the purpose of this review, we have called the first path "gene-based immu‐ notherapy", meaning that plasmid DNA does not encode a known autoantigen, and the second path "DNA vaccine immunotherapy" meaning that a beta cell autoantigen is encoded by plasmid DNA (Figure 1). In other words, gene-based immunotherapy relies on the inherent function of a product encoded by plasmid DNA that can in turn affect cell function. In contrast, DNA vaccine immunotherapy relies on the tolerogenic immune response induced directly by the autoantigen after its processing by immune cells. As we shall see, some DNA vaccine immunotherapies have also a gene-based immunotherapy component that acts as a molecular adjuvant to promote tolerogenic immune responses. Nevertheless, in all cases synthesis of the molecule encoded by plasmid DNA, which is almost always a protein, starts after delivery of

Plasmid DNA has several notable advantages compared to other vectors and therapeutic molecules. For example, it consists of relatively low molecular weight circles of double stranded DNA that can be readily isolated from bacteria in a generic and cost-effective manner. In addition, plasmid DNA permits rapid turnaround when developing new candidate products, refrigeration-free storage, and synthesis over time of a chosen antigen in its native conformation. Furthermore, plasmid DNA can be given in repeat doses within short periods of time without inducing an immune response to vector and other side effects. This is in contrast with viral-based vaccines that can induce immune responses to vector and protein/

Plasmid DNA can be delivered either as "naked" DNA or packaged into molecular scaffolds like liposomes, cationic lipids, virosomes, and polymer-based nano- and micro-particles to increase efficacy of delivery to cells [8]. Although they may sometime have pro-inflammatory properties that can be detrimental to controlling inflammation, these particles have been used to induce immune tolerance with plasmid DNA. For example, chitosan-DNA nanoparticles

Indeed, route of delivery can play a significant role in the type and strength of immune responses induced by DNA vaccines in animal models [10, 11]. In humans, two microgram of

encoding an ovalbumin antigen are tolerogenic when delivered orally [9].

**Figure 1. Plasmid DNA Immunotherapy for Type 1 Diabetes.** Plasmid DNA immunotherapy for type 1 diabetes can be divided into two categories: DNA vaccines and gene therapies. Plasmid DNA can be delivered using different routes, for example, the intradermal and intramuscular routes. The therapies can induce tolerogenic dendritic cells (DC), T regulatory cells (Treg), and death/anergy of T effector cells (Teff) which are the main cause of pancreatic islet destruction.

DNA vaccination was originally investigated to induce immunogenic responses to pathogens and cancer cells, but it has increasingly been applied to the induction of immune tolerance for treatment of autoimmune diseases like type 1 diabetes. DNA vaccines and other gene-based vaccines belong to a third generation of vaccines after live and attenuated whole organism vaccine and recombinant protein vaccines. These vaccines can be used to either prevent (prophylactic vaccine) or treat (therapeutic vaccine) disease depending on their potency, in which prevention is generally easier to achieve than treatment. Recent reports of beneficial results in different clinical trials using delivery of autoantigens indicate that DNA vaccination is reaching a stage where we are likely to see accelerated development of a therapeutic future for vaccines targeting a variety of autoimmune diseases. In the case of type 1 diabetes, early results using a DNA vaccine encoding insulin have shown promise in humans. In addition, DNA vaccines encoding human heat shock protein 60 and glutamic acid decarboxylase 65 have also shown efficacy in preclinical trials and are reviewed in this chapter.

the severity of insulitis at 13 weeks of age was not improved, the incidence of diabetes was markedly reduced to 50% at 35 week old compared to 90% with control mice. These results show that the progression of autoimmune disease in mice can effectively be suppressed by intramuscular DNA injection coding for anti-inflammatory cytokines alone. When the same interleukin-10 encoded plasmid DNA was combined with a cationic polymeric carrier, systemic administration could reduce the severity of insulitis in NOD mice markedly (15.7%) compared with that of naked DNA injection (34.5%) and non-treated controls (90.9%),

DNA Immunotherapies for Type 1 Diabetes http://dx.doi.org/10.5772/55727 537

Another report investigating the immune effects of interleukin-10 DNA showed that systemic intramuscular administration of 200 microgram interleukin-10 plasmid DNA could alleviate blood glucose and insulitis in a streptozotocin induced diabetic mouse model up to day 28 post injection [24]. In this model, pancreatic interleukin-1b and tumor necrotic factor-alpha gene expression, serum interferon-gamma concentration, and the numbers of CD4+ and

 lymphocytes were decreased on day 28. A similar interleukin-10 construct was modified by introducing nuclear factor kappa-B (NF-kB) binding sites into plasmid DNA to facilitate nuclear transport of the plasmid after delivery into the cell [25]. A single injection of 50 microgram of the plasmid using polyethylenimine as a gene carrier in 5 week old mice reduced the degree of insulitis and serum glucose levels in 100% of mice compared to 40% of the control mice at 32 weeks of age. These results illustrate how plasmid DNA can be easily modified in

As mentioned previously, nanoparticle technology has been used to condense plasmid DNA into nanometer-size complexes to improve delivery. An interleukin-10 encoding plasmid DNA was assembled into a cationic nanoparticle complex, and a single dose of 50 microgram DNA was delivered intramuscularly into streptozotocin-induced diabetic mice [26]. Animals showed higher serum levels of interleukin-10, suppression of interferon-gamma level, reduction of islet insulitis, and lower blood glucose levels compared to those treated with interleukin-10 plasmid alone or the nanoparticle alone up to week 6 post injection. Histology of muscle showed that nanoparticles were biocompatible and did not cause a chronic inflam‐

In addition to their use alone, delivery of both interleukin-4 and interleukin-10 DNA has also been investigated. Combined delivery into nonobese diabetic mice of the two plasmid DNA constructs encoding interleukin-4 and interleukin-10 (25 μg each) was done intravenously using a degradable, cationic polymeric carrier, poly (alpha-(4-aminobutyl)-L-glycolic acid) [27]. Overexpression of the two cytokine messenger RNAs was confirmed in the liver of mice 5 days after delivery. Six weeks after injection, 75% of observed islets were intact compared with less than 3% in the control group, and development of diabetes was prevented in 75% of treated animals at 30 weeks of age, compared to 20% in control mice receiving plasmid DNA coding for a single cytokine or vector control alone. The results indicated that the interleukin-4 and interleukin-10 plasmid DNAs had synergistic effects on the prevention of autoimmune diabetes. A report from the same research group showed that a 'chimeric' plasmid expressing both of the interleukin-4 and interleukin-10 under controls of two CMV promoters could also

suggesting an increased protective effect of the polymeric carrier in vivo [23].

a generic manner to improve therapeutic efficacy.

reduce insulitis in the same system [28].

CD8+

matory response.

In contrast to DNA vaccine immunotherapy, gene-based immunotherapy involves delivery of genetic material by a plasmid vector into a cell, tissue or organ with the aim of improving the clinical status using the function of the encoded product, instead of its properties as an antigen. Gene-based immunotherapy includes delivery of anti-inflammatory cytokines, chemokines, and other factors to modulate the activity of immune cells [16, 17].
