**2. Gene-based immunotherapies**

Several pre-clinical trials have used plasmid DNA-based gene therapies in experimental models of autoimmune type 1 diabetes. These strategies involve plasmid DNA designed to weaken pre-existing beta-cell autoimmunity through delivery of anti-inflammatory cytokines, chemokines, and other immune cell manipulating agents. The goal of these therapies is to reduce clinical symptoms and autoimmune outcome.

### **2.1. Cytokine gene therapies**

Cytokine gene therapies are strategies that use engineered plasmid DNA to produce thera‐ peutic immune cytokines, which are a group of immune active molecules secreted by different cells of the body. Some of these cytokines are considered beneficial for the suppression of autoimmunity, and thus are applied to disease models to reduce clinical symptoms and improve therapeutic effects. Studies of animals with spontaneous autoimmune diabetes have revealed that an important group of autoreactive T cells that mediates islet beta-cell destruction belongs to the T helper-1 type effector cell subset, and produces cytokines like interleukin-2 and interferon-gamma. On the other hand, regulatory T cells that control effector cells can secrete interleukin-4, interleukin-10, and transforming growth factor-beta.

One of the earliest applications of cytokine-engineered plasmid DNA was gene-gun delivery of murine interleukin-4 to prevent spontaneous type 1 diabetes [18]. The plasmid DNA was delivered as three times two microgram within 4 weeks into 3-week-old nonobese diabetic mice, which is the animal model system closest to human type 1 diabetes. Type 1 diabetes incidence was reduced from 90% in controls to 20% at 34 weeks of age, and was associated with T helper-2 type immune responses in the periphery and pancreas of mice. Two other reports have shown that systemic delivery of plasmid DNA constructs coding for interleukin-4 can prevent insulitis, which is an inflammatory sign of immune cell infiltrating pancreatic islets in nonobese diabetic mice [19, 20]. However, contradictory results have been reported. For example, a report indicates that intramuscular electroporation delivery of 50 μg plasmid DNA encoding interleukin-4 accelerated spontaneous type 1 diabetes in nonobese diabetic mice [21].

In addition to interleukin-4, interleukin-10-encoded plasmid DNA alone was also tested for its diabetic suppressive effects. The plasmid DNA was delivered intramuscularly twice for a total of 200 microgram into 3 and 5 week old female nonobese diabetic mice [22]. Although 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%), suggesting an increased protective effect of the polymeric carrier in vivo [23].

DNA vaccines encoding human heat shock protein 60 and glutamic acid decarboxylase 65 have

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,

Several pre-clinical trials have used plasmid DNA-based gene therapies in experimental models of autoimmune type 1 diabetes. These strategies involve plasmid DNA designed to weaken pre-existing beta-cell autoimmunity through delivery of anti-inflammatory cytokines, chemokines, and other immune cell manipulating agents. The goal of these therapies is to

Cytokine gene therapies are strategies that use engineered plasmid DNA to produce thera‐ peutic immune cytokines, which are a group of immune active molecules secreted by different cells of the body. Some of these cytokines are considered beneficial for the suppression of autoimmunity, and thus are applied to disease models to reduce clinical symptoms and improve therapeutic effects. Studies of animals with spontaneous autoimmune diabetes have revealed that an important group of autoreactive T cells that mediates islet beta-cell destruction belongs to the T helper-1 type effector cell subset, and produces cytokines like interleukin-2 and interferon-gamma. On the other hand, regulatory T cells that control effector cells can

One of the earliest applications of cytokine-engineered plasmid DNA was gene-gun delivery of murine interleukin-4 to prevent spontaneous type 1 diabetes [18]. The plasmid DNA was delivered as three times two microgram within 4 weeks into 3-week-old nonobese diabetic mice, which is the animal model system closest to human type 1 diabetes. Type 1 diabetes incidence was reduced from 90% in controls to 20% at 34 weeks of age, and was associated with T helper-2 type immune responses in the periphery and pancreas of mice. Two other reports have shown that systemic delivery of plasmid DNA constructs coding for interleukin-4 can prevent insulitis, which is an inflammatory sign of immune cell infiltrating pancreatic islets in nonobese diabetic mice [19, 20]. However, contradictory results have been reported. For example, a report indicates that intramuscular electroporation delivery of 50 μg plasmid DNA encoding interleukin-4 accelerated spontaneous type 1 diabetes in nonobese diabetic mice [21].

In addition to interleukin-4, interleukin-10-encoded plasmid DNA alone was also tested for its diabetic suppressive effects. The plasmid DNA was delivered intramuscularly twice for a total of 200 microgram into 3 and 5 week old female nonobese diabetic mice [22]. Although

secrete interleukin-4, interleukin-10, and transforming growth factor-beta.

also shown efficacy in preclinical trials and are reviewed in this chapter.

**2. Gene-based immunotherapies**

**2.1. Cytokine gene therapies**

536 Type 1 Diabetes

reduce clinical symptoms and autoimmune outcome.

chemokines, and other factors to modulate the activity of immune cells [16, 17].

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 CD8+ 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 a generic manner to improve therapeutic efficacy.

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‐ matory response.

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 reduce insulitis in the same system [28].

More recently, a research group also reported packaging plasmid DNA constructs coding for interleukin-4 and interleukin-10 into cationic nanomicelles to prevent type 1 diabetes [29]. A single intramuscular dose of 50 microgram of the complex reduced levels of blood glucose and insulitis up to 6-week post delivery in 5-week-old streptozotocin-induced diabetic mouse. It was found that levels of diabetes-associated cytokines like tumor necrotic factor-alpha and interferon-gamma were also reduced, which suggested suppression T helper-1 effector cells by the cytokine/cationic nanomicelle complex therapy. Notably, plasmid DNA coding for interleukin-4 and interleukin-10 has also been used as adjuvant to promote the therapeutic effect of DNA vaccines in a murine model for type 1 diabetes, which will describe later in this chapter in the 'Glutamic Acid Decarboxylase DNA Vaccines' section.

diabetes. Chemokine gene therapies for type 1 diabetes use anti-inflammatory chemokines as

DNA Immunotherapies for Type 1 Diabetes http://dx.doi.org/10.5772/55727 539

With regard to blocking strategies, CXC ligand-10 is an example of a chemokine associated with the pathogenesis of various T helper-1 dominant responses involved in autoimmune diseases, e.g., experimental autoimmune encephalomyelitis, rheumatoid arthritis, and type 1 diabetes. It was found that, in type 1 diabetic adult patients, elevated levels of serum CXC ligand-10 are associated with high-risk of disease in latent diabetic subjects [39]. This finding was translated in animal models where blockade or neutralization of the CXC ligand-10 can prevent type 1 diabetes in nonobese diabetic mice [40]. In virus-induced diabetic mice, virus infection results in rapid and differential expression of CXC receptor-3 and CXC ligand-10, which plays a dominant role in programming the ensuing autoimmune disease [41]. The blockade of CXC ligand-10 by using anti CXC ligand-10 monoclonal antibodies successfully aborts severity of antigen-specific injury of pancreatic beta cells and abrogates type 1 diabetes. Mechanistically, the blockade impedes the expansion of peripheral antigen-specific T effector cells and hinders their migration into the pancreas. A similar effect of the antibodies was

Based on these reports, plasmid DNA encoding the CXC ligand-10 was constructed to induce production of anti-CXC ligand-10 antibodies in the host [42]. The aim was to avoid side-effects associated with direct injection of antibodies. Intramuscular electroporation of 100 microgram of the plasmid DNA at 4 and 6 weeks of age induced synthesis of anti CXC ligand-10 antibodies *in vivo*, and suppressed the incidence of spontaneous diabetes which went from 75% in control mice down to 25% in treated mice at 30 weeks of age. Although this treatment did not inhibit insulitis or alter the immunological response, it enhanced the proliferation of pancreatic beta

A subsequent report from the same research group showed that combining complete Freund's adjuvant with plasmid DNA encoding the CXC ligand-10 could reverse diabetes [43]. Intra‐ muscular electroporation combined with complete Freund's adjuvant was used to treat newonset female nonobese diabetic mice with blood glucose levels higher than 250 mg/dL. Diabetes incidence was reduced from 70% in control mice to 20% in treated mice 10 weeks after plasmid DNA delivery. In contrast, mice receiving complete Freund's adjuvant and control plasmid DNA did not show disease reversal. In mice that were treated successfully, residual beta-cell mass was significantly increased, and some beta-cells were in a proliferative state. Although systemic cytokine profiles were unaffected, the frequency of regulatory T cells expressing CXC receptor-3 was significantly increased in local pancreatic lesions and possibly associated with

Another research group found that intra-pancreatic CC ligand-4 levels are increased in a model of diabetes protection by interleukin-4 treatment in female nonobese diabetic mice [44]. The protective effect of CC ligand-4 was confirmed by abrogation of diabetes suppression after injection of anti-CC ligand-4 antibodies [45]. These result led to studies using CC ligand-4 encoded plasmid DNA therapy which showed that gene-gun delivery of 1 microgram of the plasmid DNA protects against type 1 diabetes in NOD mice, with diabetes rates reduced from 75% in control mice to 30% at 35 weeks of age when treated weekly from week 3 to 14, and

confirmed in a cyclophosphamide accelerated model of type 1 diabetes [40].

cells and resulted in an increase of beta-cell mass.

the regulation of anti-islet autoimmunity.

well as inhibitors of pro-inflammatory chemokine binding.

In addition, a number of studies have reported that injection of plasmid DNA coding for cytokines normally considered pro-inflammatory can prevent diabetes. These results reflect the multifaceted role of cytokines on immune response [30], which could be dependent on dosage and time of administration. For example, administration of interleukin-18, also known as interferon-gamma inducing factor, can prevent diabetes in NOD mice [31, 32]. However, it was also shown that intramuscular electroporation of 2 x 100 microgram plasmid DNA coding for interleukin-18 into 4-6-week-old nonobese diabetic mice aggravates diabetes [33]. Another report showed that intraperitoneal administration of 30 microgram of plasmid DNA encoding interferon-gamma promotes insulitis in reovirus type-2 induced diabetic mice compared to controls [34]. This is in contrast with another report showing that injection of interleukin-12 induces interferon-gamma that prevents diabetes in NOD mice [35]. Together, these results suggest that there is risk associated with direct delivery of cytokines for the treatment of type 1 diabetes. This possibility is suggested by a phase I clinical trial where new onset patients with type 1 diabetes received a combination treatment of interleukin-2 and the immunosup‐ pressant rapamycin. The treatment had shown efficacy in preventing disease in mice, but it accelerated disease in humans [2].

#### **2.2. Chemokine gene therapies**

Chemokines are a family of small chemotactic cytokines secreted by cells [36]. Their name is derived from their ability to induce directed chemotaxis, or directed cell migration, in responsive cells. Some chemokines are considered pro-inflammatory and can be induced during an immune response to recruit cells of the immune system to a site of infection, while others are considered homeostatic and are involved in controlling the migration of cells during normal tissue maintenance and inhibiting abnormal inflammation like pathological autoim‐ mune response.

Chemokines are involved in pathogenesis of autoimmune disease because they can selectively recruit various subsets of immune lymphocytes [37, 38]. Based on structural motifs near their N-terminal cysteine residue [C], chemokines are divided into four subfamilies, termed CXC, CX3C, C, and CC. The function of chemokines is modulated by the type of chemokine receptors they bind to as ligands on the surface of cells, and studies have shown that chemokines and chemokine receptors are involved in the pathogenesis of autoimmune diseases like type 1 diabetes. Chemokine gene therapies for type 1 diabetes use anti-inflammatory chemokines as well as inhibitors of pro-inflammatory chemokine binding.

More recently, a research group also reported packaging plasmid DNA constructs coding for interleukin-4 and interleukin-10 into cationic nanomicelles to prevent type 1 diabetes [29]. A single intramuscular dose of 50 microgram of the complex reduced levels of blood glucose and insulitis up to 6-week post delivery in 5-week-old streptozotocin-induced diabetic mouse. It was found that levels of diabetes-associated cytokines like tumor necrotic factor-alpha and interferon-gamma were also reduced, which suggested suppression T helper-1 effector cells by the cytokine/cationic nanomicelle complex therapy. Notably, plasmid DNA coding for interleukin-4 and interleukin-10 has also been used as adjuvant to promote the therapeutic effect of DNA vaccines in a murine model for type 1 diabetes, which will describe later in this

In addition, a number of studies have reported that injection of plasmid DNA coding for cytokines normally considered pro-inflammatory can prevent diabetes. These results reflect the multifaceted role of cytokines on immune response [30], which could be dependent on dosage and time of administration. For example, administration of interleukin-18, also known as interferon-gamma inducing factor, can prevent diabetes in NOD mice [31, 32]. However, it was also shown that intramuscular electroporation of 2 x 100 microgram plasmid DNA coding for interleukin-18 into 4-6-week-old nonobese diabetic mice aggravates diabetes [33]. Another report showed that intraperitoneal administration of 30 microgram of plasmid DNA encoding interferon-gamma promotes insulitis in reovirus type-2 induced diabetic mice compared to controls [34]. This is in contrast with another report showing that injection of interleukin-12 induces interferon-gamma that prevents diabetes in NOD mice [35]. Together, these results suggest that there is risk associated with direct delivery of cytokines for the treatment of type 1 diabetes. This possibility is suggested by a phase I clinical trial where new onset patients with type 1 diabetes received a combination treatment of interleukin-2 and the immunosup‐ pressant rapamycin. The treatment had shown efficacy in preventing disease in mice, but it

Chemokines are a family of small chemotactic cytokines secreted by cells [36]. Their name is derived from their ability to induce directed chemotaxis, or directed cell migration, in responsive cells. Some chemokines are considered pro-inflammatory and can be induced during an immune response to recruit cells of the immune system to a site of infection, while others are considered homeostatic and are involved in controlling the migration of cells during normal tissue maintenance and inhibiting abnormal inflammation like pathological autoim‐

Chemokines are involved in pathogenesis of autoimmune disease because they can selectively recruit various subsets of immune lymphocytes [37, 38]. Based on structural motifs near their N-terminal cysteine residue [C], chemokines are divided into four subfamilies, termed CXC, CX3C, C, and CC. The function of chemokines is modulated by the type of chemokine receptors they bind to as ligands on the surface of cells, and studies have shown that chemokines and chemokine receptors are involved in the pathogenesis of autoimmune diseases like type 1

chapter in the 'Glutamic Acid Decarboxylase DNA Vaccines' section.

accelerated disease in humans [2].

**2.2. Chemokine gene therapies**

mune response.

538 Type 1 Diabetes

With regard to blocking strategies, CXC ligand-10 is an example of a chemokine associated with the pathogenesis of various T helper-1 dominant responses involved in autoimmune diseases, e.g., experimental autoimmune encephalomyelitis, rheumatoid arthritis, and type 1 diabetes. It was found that, in type 1 diabetic adult patients, elevated levels of serum CXC ligand-10 are associated with high-risk of disease in latent diabetic subjects [39]. This finding was translated in animal models where blockade or neutralization of the CXC ligand-10 can prevent type 1 diabetes in nonobese diabetic mice [40]. In virus-induced diabetic mice, virus infection results in rapid and differential expression of CXC receptor-3 and CXC ligand-10, which plays a dominant role in programming the ensuing autoimmune disease [41]. The blockade of CXC ligand-10 by using anti CXC ligand-10 monoclonal antibodies successfully aborts severity of antigen-specific injury of pancreatic beta cells and abrogates type 1 diabetes. Mechanistically, the blockade impedes the expansion of peripheral antigen-specific T effector cells and hinders their migration into the pancreas. A similar effect of the antibodies was confirmed in a cyclophosphamide accelerated model of type 1 diabetes [40].

Based on these reports, plasmid DNA encoding the CXC ligand-10 was constructed to induce production of anti-CXC ligand-10 antibodies in the host [42]. The aim was to avoid side-effects associated with direct injection of antibodies. Intramuscular electroporation of 100 microgram of the plasmid DNA at 4 and 6 weeks of age induced synthesis of anti CXC ligand-10 antibodies *in vivo*, and suppressed the incidence of spontaneous diabetes which went from 75% in control mice down to 25% in treated mice at 30 weeks of age. Although this treatment did not inhibit insulitis or alter the immunological response, it enhanced the proliferation of pancreatic beta cells and resulted in an increase of beta-cell mass.

A subsequent report from the same research group showed that combining complete Freund's adjuvant with plasmid DNA encoding the CXC ligand-10 could reverse diabetes [43]. Intra‐ muscular electroporation combined with complete Freund's adjuvant was used to treat newonset female nonobese diabetic mice with blood glucose levels higher than 250 mg/dL. Diabetes incidence was reduced from 70% in control mice to 20% in treated mice 10 weeks after plasmid DNA delivery. In contrast, mice receiving complete Freund's adjuvant and control plasmid DNA did not show disease reversal. In mice that were treated successfully, residual beta-cell mass was significantly increased, and some beta-cells were in a proliferative state. Although systemic cytokine profiles were unaffected, the frequency of regulatory T cells expressing CXC receptor-3 was significantly increased in local pancreatic lesions and possibly associated with the regulation of anti-islet autoimmunity.

Another research group found that intra-pancreatic CC ligand-4 levels are increased in a model of diabetes protection by interleukin-4 treatment in female nonobese diabetic mice [44]. The protective effect of CC ligand-4 was confirmed by abrogation of diabetes suppression after injection of anti-CC ligand-4 antibodies [45]. These result led to studies using CC ligand-4 encoded plasmid DNA therapy which showed that gene-gun delivery of 1 microgram of the plasmid DNA protects against type 1 diabetes in NOD mice, with diabetes rates reduced from 75% in control mice to 30% at 35 weeks of age when treated weekly from week 3 to 14, and from 80% in control mice down to 30% when treated weekly from week 9 to 14 [45]. Data also indicated that plasmid DNA delivery could both prevent and treat type 1 diabetes. This protection was associated with a T helper-2-like response in the spleen and pancreas, decreased recruitment of activated CD8 T cells to islets accompanied by diminished CC receptor-5 expression on CD8 T cells, and increased regulatory T cell activity in the draining pancreatic lymph nodes.

of 100 microgram of plasmid DNA coding for decoy receptor 9 into nonobese diabetic mice was reported to reduce diabetes incidence from 90% in controls to 30% when treated at 4 weeks of age, 45% (started at 7 week old), and 70% (as Fc-fusion form, started at 12 week old) in 35 week-old female nonobese diabetic mice [50]. Treated mice showed less splenocyte prolifera‐ tion and adoptive transfer of the cells ameliorated diabetes. Treated NOD-T1/2 mice had reduced T helper-1, T helper-17, and increased T helper-2 immune responses *in vivo*. Data also indicated that immune modulation by decoy receptor 3 may have been the result of differen‐ tiation and maturation of dendritic cells that subsequently regulated T effector differentiation

DNA Immunotherapies for Type 1 Diabetes http://dx.doi.org/10.5772/55727 541

Cell migration is another process that plays a role in pancreatic beta cell destruction. In this regard, plasmid DNA coding for CD44, which is a protein associated with cell migration and delivery of apoptotic signals by inflammatory cells, was investigated for the suppression of diabetes. It was found that subcutaneous implants of a silicone tube filled with wound dressing sponge carrying CD44 encoded plasmid DNA could attenuate diabetes in a transfer model [51]. Diabetes was induced in male nonobese diabetic mice by transfer of diabetogenic splenocytes from female diabetic mice and was reduced from 90% in controls to 20-30% 12 weeks after two implants. Here the mechanism of treatment was not thought to be strictly a gene therapy effect, but rather induction of anti-CD44 antibodies that inhibited CD44 function. An increasing body of evidence points to a possible relationship between the central nervous system and diabetes [52]. For example, the pancreatic autoantigen glutamic acid decarboxylase is an enzyme responsible for synthesis of the neurotransmitter gamma-aminobutyric acid (GABA) in the peripheral and central nervous system. Notably, at least two neurotransmitterrelated peptides have been used successfully as plasmid DNA immunotherapies for type 1

The first peptide is calcitonin gene-related peptide (CGRP), which has been found to play an important role in the regulation of T lymphocytes and in protecting cells from reactive oxygen species. It was found that a single injection of 200 microgram plasmid DNA encoding the peptide delivered intramuscularly using electroporation could significantly ameliorate hyperglycemia and insulin deficiency [53]. The treatment decreased diabetes incidence from 73% in controls to 23% at 28 days post delivery in a streptozotocin-induced diabetic model. The gene transfer also significantly inhibited T cell proliferation and secretion of the T helper-1 cytokine interferon-gamma, increased the levels of the T helper-2 cytokine interleukin-10, but had no effect on interleukin-4 and transforming growth factor-beta secretion. Therefore, calcitonin gene-related peptide gene transfer appears to ameliorate streptozotocin-induced

The second peptide is named vasoactive intestinal peptide (VIP) and functions as a neuromodulator and neurotransmitter [54]. The peptide is a potent vasodilator that regulates smooth muscle activity, epithelial cell secretion, and blood flow in the gastrointes‐ tinal tract. Importantly, a body of evidence points to a significant role of vasoactive intestinal polypeptide in regulating immune responses. The peptide acts as a potent endogenous anti-inflammatory molecule and promotes the activity of T regulatory cells, which makes it a promising candidate for the treatments of inflammatory and autoim‐

and function.

diabetes.

diabetes through immune deviation.

To summarize, plasmid DNA encoding CC ligand-4 and CXC ligand-10 have been tested for their type 1 diabetic suppressive effects in spontaneous diabetic mouse models. Diabetes suppression is associated with decreased CD8 T lymphocyte activity and increased CD4 T regulatory cell activity. These results suggest a possible clinical application of chemokine ligand gene therapies, although they are anticipated to have possible side effects associated with systemic immunotherapies.

#### **2.3. Other cell-manipulating gene therapies**

Several immune cell populations have deficiencies in type 1 diabetes, such as CD4 T lympho‐ cytes, CD8 T lymphocytes, B lymphocytes, dendritic cells, macrophages, and NK cells in both nonobese diabetic mice and human patients [46, 47]. The goal of cell-manipulating gene therapy is to increase the diabetic suppressive function of cells like T regulatory or T helper-2 lymphocytes, which are considered important not only for therapeutic purposes, but also for playing a determining role in the development of type 1 diabetes.

As mentioned at the beginning of this chapter, type 1 diabetes is a T helper-1-mediated autoimmune disease and strategies suppressing the function of these cells can be expected to have an impact on disease progression. One of these strategies is the delivery of galectin-9, a carbohydrate-binding protein that regulates T helper-1 cells and induces their apoptosis through the galectin-9 receptor. Apoptosis, or programmed-cell-death, is a constantly ongoing process in steady state *in vivo* and helps maintain tissue and immune homeostasis. Mice treated with plasmid DNA coding for galectin-9 were significantly protected from diabetes: intrave‐ nous delivery of 2 x 100 microgram bi-weekly protected 85% of mice from diabetes versus 55% in controls [48]. Analysis of immune responses showed that the T helper-1 cell population was markedly decreased in spleen, pancreas, and pancreatic lymph nodes of treated NOD-T1/2 double transgenic diabetic mice, indicating a suppressive role of galectin-9 on pathogenic T helper-1 cells. Splenocytes from treated mice were also less responsive to mitogenic stimula‐ tion than splenocytes from the control group. Data indicated that galectin-9 DNA may downregulate T helper-1 immune response in diabetic mice and could be used as a therapeutic agent in autoimmune diabetes.

In contrast with galectin-9, decoy receptor 3 inhibits apoptosis. The membrane protein is a member of the tumor necrosis factor receptor superfamily, and regulates immune responses by neutralizing apoptotic signals transmitted through CD95 (Fas receptor), lymphotoxin betareceptor, and death receptor 3 on target cells. As a result, transgenic expression of decoy receptor 3 in pancreatic beta cells protects nonobese diabetic mice from autoimmune diabetes [49]. When decoy receptor 3 is delivered systemically as plasmid DNA, it inhibits insulitis and diabetes by modulating immune responses. For example, four weekly intravenous injections of 100 microgram of plasmid DNA coding for decoy receptor 9 into nonobese diabetic mice was reported to reduce diabetes incidence from 90% in controls to 30% when treated at 4 weeks of age, 45% (started at 7 week old), and 70% (as Fc-fusion form, started at 12 week old) in 35 week-old female nonobese diabetic mice [50]. Treated mice showed less splenocyte prolifera‐ tion and adoptive transfer of the cells ameliorated diabetes. Treated NOD-T1/2 mice had reduced T helper-1, T helper-17, and increased T helper-2 immune responses *in vivo*. Data also indicated that immune modulation by decoy receptor 3 may have been the result of differen‐ tiation and maturation of dendritic cells that subsequently regulated T effector differentiation and function.

from 80% in control mice down to 30% when treated weekly from week 9 to 14 [45]. Data also indicated that plasmid DNA delivery could both prevent and treat type 1 diabetes. This protection was associated with a T helper-2-like response in the spleen and pancreas, decreased recruitment of activated CD8 T cells to islets accompanied by diminished CC receptor-5 expression on CD8 T cells, and increased regulatory T cell activity in the draining pancreatic

To summarize, plasmid DNA encoding CC ligand-4 and CXC ligand-10 have been tested for their type 1 diabetic suppressive effects in spontaneous diabetic mouse models. Diabetes suppression is associated with decreased CD8 T lymphocyte activity and increased CD4 T regulatory cell activity. These results suggest a possible clinical application of chemokine ligand gene therapies, although they are anticipated to have possible side effects associated

Several immune cell populations have deficiencies in type 1 diabetes, such as CD4 T lympho‐ cytes, CD8 T lymphocytes, B lymphocytes, dendritic cells, macrophages, and NK cells in both nonobese diabetic mice and human patients [46, 47]. The goal of cell-manipulating gene therapy is to increase the diabetic suppressive function of cells like T regulatory or T helper-2 lymphocytes, which are considered important not only for therapeutic purposes, but also for

As mentioned at the beginning of this chapter, type 1 diabetes is a T helper-1-mediated autoimmune disease and strategies suppressing the function of these cells can be expected to have an impact on disease progression. One of these strategies is the delivery of galectin-9, a carbohydrate-binding protein that regulates T helper-1 cells and induces their apoptosis through the galectin-9 receptor. Apoptosis, or programmed-cell-death, is a constantly ongoing process in steady state *in vivo* and helps maintain tissue and immune homeostasis. Mice treated with plasmid DNA coding for galectin-9 were significantly protected from diabetes: intrave‐ nous delivery of 2 x 100 microgram bi-weekly protected 85% of mice from diabetes versus 55% in controls [48]. Analysis of immune responses showed that the T helper-1 cell population was markedly decreased in spleen, pancreas, and pancreatic lymph nodes of treated NOD-T1/2 double transgenic diabetic mice, indicating a suppressive role of galectin-9 on pathogenic T helper-1 cells. Splenocytes from treated mice were also less responsive to mitogenic stimula‐ tion than splenocytes from the control group. Data indicated that galectin-9 DNA may downregulate T helper-1 immune response in diabetic mice and could be used as a therapeutic

In contrast with galectin-9, decoy receptor 3 inhibits apoptosis. The membrane protein is a member of the tumor necrosis factor receptor superfamily, and regulates immune responses by neutralizing apoptotic signals transmitted through CD95 (Fas receptor), lymphotoxin betareceptor, and death receptor 3 on target cells. As a result, transgenic expression of decoy receptor 3 in pancreatic beta cells protects nonobese diabetic mice from autoimmune diabetes [49]. When decoy receptor 3 is delivered systemically as plasmid DNA, it inhibits insulitis and diabetes by modulating immune responses. For example, four weekly intravenous injections

lymph nodes.

540 Type 1 Diabetes

with systemic immunotherapies.

agent in autoimmune diabetes.

**2.3. Other cell-manipulating gene therapies**

playing a determining role in the development of type 1 diabetes.

Cell migration is another process that plays a role in pancreatic beta cell destruction. In this regard, plasmid DNA coding for CD44, which is a protein associated with cell migration and delivery of apoptotic signals by inflammatory cells, was investigated for the suppression of diabetes. It was found that subcutaneous implants of a silicone tube filled with wound dressing sponge carrying CD44 encoded plasmid DNA could attenuate diabetes in a transfer model [51]. Diabetes was induced in male nonobese diabetic mice by transfer of diabetogenic splenocytes from female diabetic mice and was reduced from 90% in controls to 20-30% 12 weeks after two implants. Here the mechanism of treatment was not thought to be strictly a gene therapy effect, but rather induction of anti-CD44 antibodies that inhibited CD44 function.

An increasing body of evidence points to a possible relationship between the central nervous system and diabetes [52]. For example, the pancreatic autoantigen glutamic acid decarboxylase is an enzyme responsible for synthesis of the neurotransmitter gamma-aminobutyric acid (GABA) in the peripheral and central nervous system. Notably, at least two neurotransmitterrelated peptides have been used successfully as plasmid DNA immunotherapies for type 1 diabetes.

The first peptide is calcitonin gene-related peptide (CGRP), which has been found to play an important role in the regulation of T lymphocytes and in protecting cells from reactive oxygen species. It was found that a single injection of 200 microgram plasmid DNA encoding the peptide delivered intramuscularly using electroporation could significantly ameliorate hyperglycemia and insulin deficiency [53]. The treatment decreased diabetes incidence from 73% in controls to 23% at 28 days post delivery in a streptozotocin-induced diabetic model. The gene transfer also significantly inhibited T cell proliferation and secretion of the T helper-1 cytokine interferon-gamma, increased the levels of the T helper-2 cytokine interleukin-10, but had no effect on interleukin-4 and transforming growth factor-beta secretion. Therefore, calcitonin gene-related peptide gene transfer appears to ameliorate streptozotocin-induced diabetes through immune deviation.

The second peptide is named vasoactive intestinal peptide (VIP) and functions as a neuromodulator and neurotransmitter [54]. The peptide is a potent vasodilator that regulates smooth muscle activity, epithelial cell secretion, and blood flow in the gastrointes‐ tinal tract. Importantly, a body of evidence points to a significant role of vasoactive intestinal polypeptide in regulating immune responses. The peptide acts as a potent endogenous anti-inflammatory molecule and promotes the activity of T regulatory cells, which makes it a promising candidate for the treatments of inflammatory and autoim‐

mune diseases, such as septic shock, arthritis, multiple sclerosis, Crohn disease, and autoimmune diabetes [55, 56]. For example, a single intramuscular injection of 300 microgram of vasoactive intestinal polypeptide-encoding plasmid DNA significantly reduced the incidence of cyclophosphamide accelerated diabetes in female nonobese diabetic mice, from 70% in control to 30% on day 33 post delivery in 8-10-week-old mice [57]. A subsequent report in a different model system indicated that injection of the peptide could activate FoxP3+ CD4+ CD25high T regulatory cells and protect against acute graft-versushost disease in a mouse model of allogeneic bone marrow transplantation [58].

**Categories Encoded Products Immune Responses Trials References**

Cytokines IL-4 T helper-2 Preclinical [18-21]

Chemokines C-C Motif Ligand 4 T helper-2, Tregs Preclinical [45]

Cellular Galectin-9 T helper-2 Preclinical [48] Manipulations Decoy Receptor 3 T helper-2, DCs Preclinical [50]

IL-4 + IL-10 as

Proinsulin DNA + Peptide CD25-

GAD65-Ig Fc +

Proinsulin ?

Proinsulin + Anti-CD3 T helper-2, CD25+

**Table 1. Plasmid DNA Based Immunotherapies for Type 1 Diabetes.** The table summarizes plasmid DNA based immunotherapies under two categories: Gene therapies and DNA vaccines. Immunotherapies are listed according to their category, type of immune response and trial. Abbreviation: IL, interleukin; CD, cluster of differentiation; PRPIII, pancreatic regenerating protein III; CGRP, calcitonin gene-related peptide; VIP, vasoactive intestinal polypeptide; HSP, heat shock protein; GAD, glutamic acid decarboxylase. IA, insulinoma associated protein; Tregs, T regulatory cells; DCs:

IL-10 T helper-2 Preclinical [22-26]

Nanoparticle T helper-2 Preclinical [27-29]

CD44 Antibodies Preclinical [51] CGRP T helper-2 Preclinical [53] VIP Tregs Preclinical [57]

Insulin B chain T helper-2 Preclinical [60,67] Insulin B Chain + IL-4 T helper-2 Preclinical [61] Proinsulin + Anti-CD154 CD25+ Tregs Preclinical [62]

Proinsulin + PRP III CD25+ Tregs Preclinical [64] Pre-proinsulin + B7-1wa ? Preclinical [65]

HSP 60 T helper-2 PreClinical [79] HSP 65 T helper-2, Tregs Preclinical [80,81] Secreted GAD65, GAD65 T helper-2 Preclinical [84,85,88,92]

IL-4/IL-10/IL-4 & IL-10 T helper-2 Preclinical [86,87] GAD65 + Anti-CD3 Tregs Preclinical [94] Secreted GAD65 + BAX Tregs, DCs Preclinical [97-99] IA-2 + IL-4 Tregs? Preclinical [101] GPI + lysosome nTregs Preclinical [102]

Tregs

Tregs Preclinical [63]

Preclinical,

Clinical [15,66,68]

DNA Immunotherapies for Type 1 Diabetes http://dx.doi.org/10.5772/55727 543

Preclinical [71]

C-X-C Motif Ligand 10 Tregs Preclinical [42,43]

Gene Therapies

DNA Vaccines

dendritic cells.

#### **2.4. Summary of Section 2**

Section 1 covers plasmid DNA encoding small protein molecules like cytokines, chemokines, peptides and other immune cell-manipulating agents with therapeutic effects on preclinical type 1 diabetes (Table 1). These approaches belong to systemic treatments and inevitably bear the risks associated with nonspecific immune suppression and chronic complications resulting from interference with the host immune system. Nonetheless, if used as adjuvants or supple‐ ments to pancreatic autoantigen-targeting therapies like DNA vaccines, these approaches could be used selectively in DNA-based combination therapies. We provide examples of such approaches in Section 2 of this chapter.
