U.S. flag

An official website of the United States government

NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

Cover of Madame Curie Bioscience Database

Madame Curie Bioscience Database [Internet].

Show details

Immunogene Therapy with Nonviral Vectors

, , and .

Author Information and Affiliations

Introduction

The majority of gene therapy studies have been performed with viral vectors that present important limitations in terms of immunogenicity and pathogenicity. Nonviral (usually plasmid-based) gene therapy is not hampered by these limitations and, although gene transfer is generally less efficient, it has been successfully employed in the prevention or treatment of several experimental autoimmune diseases.1-12 Gene transfer of naked DNA can be enhanced by several methods and, at least for some applications, can now rival or even surpass viral gene transfer. Indeed, in animal models of disease, nonviral methods are effective at delivering cDNA encoding regulatory cytokines such as IL-10 or transforming growth factor β1 (TGF-β1), which exert many anti-inflammatory effects and promote the activity of regulatory T cells (Tr). This approach is also effective for the administration of cytokine inhibitors such as IL-1 receptor antagonist (IL-1Ra), soluble interferon gamma (IFNγ) receptor (IFNγR)/IgG-Fc fusion protein, or TNFα receptor (TNFR).9-12 Furthermore, in vivo transfer of nucleic acid segments (or plasmid-based delivery of these molecules), such as cytosine-phosphate-guanine (CpG)-containing oligodeoxynucleotides (ODNs) or small inhibitory RNA (siRNA), is highly promising in the therapy of conditions as diverse as autoimmune diseases, other inflammatory disorders, allergy, infectious diseases and cancer. In this chapter, we will focus primarily on nonviral gene therapy of autoimmune diseases and other inflammatory disorders, although applications to other diseases will be mentioned when relevant.

The design of effective immunotherapies must include determination of the immune mechanisms directly responsible for inflammatory tissue injury. In this respect, significant pathology can be attributed to the inflammatory cytokines IL-1, TNFα, IL-12 and IFNγ, or molecularly related cytokines.13-20 Moreover, any combination of these cytokines is likely to be even more injurious than each component alone. IL-1, TNFα and IL-12 are produced principally by macrophages and dendritic cells, whereas IFNγ is produced by T-helper (Th) type 1 (Th1) cells, cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. For example, in nonobese diabetic (NOD) mice with autoimmune diabetes (type 1 diabetes [T1D]), mononuclear infiltration of the islets of Langerhans (insulitis) is associated with local IL-12 and IFNγ production reflecting, at least in part, a Th1-dependent reaction.19,20 Similarly, Th1-mediated inflammatory pathology has been observed in experimental autoimmune encephalomyelitis (EAE), and this disease can be passively transferred with autoagressive Th1 clones reacting to either myelin basic protein (MBP), proteolipid protein (PLP), or other central nervous system (CNS) antigens.21,22

Clinically, the neutralization of TNFα with monoclonal antibodies (mAbs) or soluble receptors has proven effective in the treatment of rheumatoid arthritis (RA),14-17 and this represents one of the most effective immunotherapies designed in recent years. Consequently, gene therapists have attempted to ameliorate autoimmune diseases by neutralizing the activity of inflammatory cytokines. Obviously, as shown in RA, this can also be accomplished with protein drugs such as mAbs or recombinant receptors. However, these proteins have to be repeatedly administered in large amounts by parenteral routes. MAbs are also subject to neutralization by the recipient's immune response, even though this can be reduced by “humanization” of the antibodies. As an alternative to direct anticytokine therapy, it is feasible to administer regulatory cytokines (e.g., IL-4, IL-10 or TGF-β) which inhibit the production of inflammatory mediators. Unfortunately, the use of cytokines as protein therapeutic agents is also markedly problematic because they are expensive to produce, have short half-lives, and frequently exert toxic effects, especially when administered as a bolus.

Gene therapy offers the possibility of eliminating or diminishing some of these problems. It permits long-term, relatively constant delivery of anti-inflammatory or immunoregulatory mediators. In the case of cytokines, this can be accomplished at low levels which are less likely to be toxic. Specific tissues can be targeted, such as the joints or the CNS. Very recent studies suggest that the production of pathogenic inflammatory mediators can be inhibited with gene-specific short (small) inhibitory RNAs (siRNAs). Furthermore, as discussed in other chapters, appropriate genes can be transduced into autoantigen-specific T cells ex vivo. These cells can then be injected into diseased animals, where they specifically infiltrate the antigen-bearing target organ, and downregulate autoimmune processes.

Nonviral Gene Therapy Vectors

Almost all the nonviral vectors employed thus far are expression plasmids, which have been designed for high expression in striated muscle cells or other cells. The construction of these vectors is quite simple and straightforward. The best plasmids carry a strong promoter (most of often the human cytomegalovirus (CMV) immediate-early enhancer promoter [IE-EP]), an intron (such as CMV intron A), a multiple cloning site for insertion of the gene of interest, and an appropriate transcriptional terminator segment.

The construction and in vivo delivery of these vectors has been extensively reviewed,8-11 and will only be briefly described here. The transfer of naked plasmid DNA following needle injection occurs more readily in skeletal muscle than in most other tissues.10,11 Furthermore, in various tissues, transfection has been enhanced or accomplished by “gene gun” delivery (usually DNA-coated gold particles propelled into cells),23 jet injection of DNA,24,25 cationic polymers such as polyethylenimine (PEI) and poly-L-lysine (PLL), and cationic liposomes.25 Recently, in vivo electroporation has been shown to be one of the most effective approaches.25-28 In addition, infusion of plasmids under pressure in veins or arteries (hydrodynamic delivery) results in the extensive transfection of cells in tissues supplied by the relevant vasculature, such as liver or muscle.27,29,30 Indeed, hydrodynamic approaches are advantageous when the transfection of very large number of cells in a tissue is desired. Although some hydrodynamic approaches are not feasible in humans, due to the large amount of fluid that is rapidly infused, modified approaches (especially on isolated limbs) appear clinically applicable.31 Interestingly, ultrasound has also been employed to enhance gene delivery,32-34 but has been less effective than either electroporation or hydrodynamic delivery. Nevertheless, various physical methods can be combined (electroporation and ultrasound or other combinations), to further improve transfection.34

Optimizing Gene Transfer

Skeletal muscle represents an advantageous target for nonviral gene therapy, as first demonstrated by Wolff and his colleagues.35,36 It accounts for 30-50% of the body weight, and is easily accessible and abundantly vascularized. Moreover, transgene expression is generally much more prolonged than in other tissues, probably because striated myocytes are nondividing, long-lived cells. In mice, we observed that protein production reaches a maximum after the injection of 50-100 μg of naked plasmid DNA per muscle.11 Without any special maneuvers to enhance transfection, 50 μg of DNA can lead to the synthesis of > 300 ng of nonsecreted reporter protein (e.g., luciferase).37 In the case of secreted proteins, serum values can range from a few picograms/ ml to > 300 ng/ml.2-5,38 Not surprisingly, several factors affect these results, including vector components (e.g., promoters, introns and terminator sequences) and the rate of protein turnover. Maximum protein levels are most frequently recorded 1 to 2 weeks after DNA administration, but the persistence of expression varies greatly depending on the antigenicity of the product and other factors. The presence of unmethylated CpG motifs (see below) in the vector has an inflammatory effect which contributes to the shut down of expression, and the inclusion of genes encoding inflammatory cytokines is likely to have a similar negative effect.

In mice, the injection of a 50 μl dose of fluorescence-labeled plasmid into the tibialis anterior muscle is followed by the rapid diffusion of DNA throughout the muscle.39 DNA is internalized by myocytes within 5 min, and over several hours by mononuclear cells (perhaps macrophages or dendritic cells) located along muscle fibers and in the draining lymph nodes. Notably, the transgene is expressed primarily by muscle cells,39 but DNA vaccination studies suggest that dendritic cells (DCs) are also transfected, particularly when electroporation is applied. Since DCs are present in small numbers in normal muscle, this is not easy to demonstrate. The mechanism by which plasmids travel from the extracellular space to the nuclei of skeletal muscle cells remains unclear. However, it may be of relevance that these cells are multinucleated and the nuclei are located peripherally, in apposition to the cell membrane.

In Vivo Electroporation

Numerous studies5,25-28,39-54 have shown that in vivo low voltage electroporation greatly augments transfection. Thus, electrogene transfer (EGT) increased reporter protein production in muscle by 100-fold or more in some studies. Electric pulses are thought to increase DNA entry into cells by creating transient pores in the cell membrane, and by promoting DNA motility (electrophoretic effect). Electroporation is a versatile approach, and has been successfully used to enhance DNA transfer into muscle (heart, skeletal),26,39-47,54 liver,50 brain,51 various tumors, testis, bladder, embryos and other tissues.31,34,35,53 This technique is an adaptation of electrochemotherapy (ECT), where in vivo electroporation (electropermeabilization) promotes entry of some anti-cancer drugs (e.g., Bleomycin) into cells, presumably due to the formation of transient pores in cell membranes.54-58 ECT has been effective in animal models and clinical trials. Furthermore, ECT and EGT have been successfully combined for anti-tumor therapy.58

To enhance intramuscular gene transfer, electrical pulses using invasive or noninvasive electrodes are applied at the site of, and shortly after, DNA injection.5,34,54 Optimally, this consists of low field strength (100-200 V/cm), relatively long (20-50 milliseconds) squarewave electric pulses, applied 6-8 times in quick succession. These values are based on our own experience, but are similar to those reported by most other authors. These low-voltage electrical pulses cause muscle damage, but it is usually mild and transient. For instance, Mathiesen43 examined muscles 3 days after injection of DNA and electroporation under various conditions, and observed regions of necrotic fibers of increasing extent with increasing cumulative pulse duration. The majority of surviving fibers expressed the reporter gene. Two weeks after electroporation the muscles appeared grossly normal. He noted the presence of muscle fibers with central nuclei, most likely indicating muscle regeneration from satellite cells.

CpG Motifs and Toll-Like Receptor 9 (Tlr9)

An important component of the plasmid is the presence of unmethylated CpG-containing immunostimulatory sequences (ISS), that can activate innate immunity by binding to TLR9 located in endocytic vesicles of APCs.59-61 Engagement of TLR9 triggers a cell signaling cascade involving sequentially myeloid differentiation primary response gene 88 (MyD88), interleukin-1 receptor activated kinase (IRAK), tumour necrosis factor receptor (TNFR)-associated factor 6 (TRAF6), and activation of NFκB.59 Cells that express TLR9, which include plasmacytoid dendritic cells (PDCs) and B cells, produce interferon a and β (IFNaβ), inflammatory cytokines such as IL-12, and chemokines.

PDCs represent a small subpopulation of cells with the ability to produce large amounts of IFNαβ,62,63 which promotes a Th1 response. It appears that the early (innate) production of IFNαβ at the beginning of an immune response stimulates CD8+ T-cell proliferation and promotes activation of NK cells. CpG-stimulated DCs produce IL-12 which activates acquired antigen-specific T cell responses. There are important inter-species differences, in that in humans only PDCs and B cells express TLR9 (although other cells respond to TLR9 engagement, presumably by indirect stimulation), while in mice cells of other phenotypes, such as monocytes, macrophages and myeloid DCs, also express this receptor.62-63 These differences are likely to influence the outcome of DNA vaccination and are highly relevant to vaccine design.

Optimal CpG motifs for activating mouse or rabbit immune cells have the general formula, purine-purine-CG-pyrimidine-pyrimidine.59-61 However, for activating human cells, and cells of several other species, the optimal motif is TCGTT and/or TCGTA. In addition, some sequences that are immediately adjacent to these short motifs can contribute to the immunostimulatory effects.59 Three classes of CpG-containing oligodeoxynucleotides (ODNs) have been described.59-61 CpG ODNs of the B-class (also called K-class) strongly stimulate B cells, promote PDC maturation, but induce only low amounts of IFNαβ. In contrast, A-class (also called D-class) ODNs strongly stimulate plasmacytoid DCs (PDCs) to secrete IFNαβ, but are poor at activating B cells. C-Class ODNs combine the properties of the A and B classes, and are very strong Th1 adjuvants. The high levels of IFNa induced by either A-class or C-class ODNs activate NK cells efficiently. Moreover, CpG ODNs promote the transition from monocytes to myeloid DCs, and contribute to DC maturation.

Not all CpG ODNs are stimulatory. Suppressive motifs have also been described and they are rich in polyG or -GC sequences, tend to be methylated, and are present in the DNA of mammals and certain viruses.60 These neutralizing motifs (CpG-N motifs) also exit in plasmids. Most DNA vaccines contain numerous CpG motifs, some of which are in an immunostimulatory context, while others are inhibitory. Thus, the ultimate effect of the plasmid DNA backbone in DNA vaccination may depend on the ratio of stimulatory and inhibitory sequences. Indeed, Klinman and his colleagues64,65 report that the immunostimulatory activity of CpG ODNs can be abrogated in vitro and in vivo by the addition of suppressive sequences. It appears that stimulatory and suppressive ODNs bind to the same cells, and suppression tends to be dominant. When both types of sequences are joined recognition proceeds in a 5' to 3' direction, such that a 5' motif can interfere with one that is located immediately downstream. Suppressive motifs interfere with the maturation of endosomal vesicles and the colocalization of CpG ODNs and TLR9 in these vesicles. Interestingly, suppressive ODNs can protect against CpG-induced lesions, such as arthritis.65

The innate immune response created by CpG ISS is desirable for DNA vaccination, since it promotes the maturation of DCs and primes T cells to respond to the relevant antigen. However, CpG motifs are detrimental in gene therapy studies. First, their nonspecific inflammatory effects might directly injure tissues,and/or confuse the interpretation of immunological studies.Second, the CMV IE-EP, and other viral promoters, are turned off by inflammatory cytokines (particularly IFNγ and TNFα).66-68 Since most plasmids carry large numbers of CpG motifs, it is not easy to eliminate them completely. Nevertherless, some recently available commercial plasmid vectors are devoid of CpG elements, even in sequences coding for reporter genes (e.g., InVivogen, San Diego, CA). This is possible because of the eight codons that contain CG, all can be substituted by at least two other codons that code for the same amino acid. Moreover, it appears that CpG motifs bind to TLR9 only in an unmethylated form. A recent study69 revealed that methylation of plasmids abrogates CpG/TLR9 interactions, while retaining vector expression. Mice inoculated with a CpG-methylated plasmid expressing a viral protein showed delayed clearance of transfected cells and failed to mount a strong immune response to the viral product. Importantly, the persistence of vector expression was increased.

An alternative approach involves deletion of most vector elements, to produce minicircles containing only, or primarily, the expression cassette.70,71 These small vectors transfect cells more efficiently, presumably because of their small size. Furthermore, they lack all the CpG sequences of the vector backbone, and retain only those that might be present in essential transcriptional elements (these can also be replaced with alternative codons). Minicircle DNA vectors are remarkable for the level and persistence of transgene expression. Indeed, minicircular DNAs lacking bacterial sequences expressed 45- and 560-fold more serum human factor IX and alpha1-antitrypsin, respectively, compared to standard plasmid DNAs transfected into mouse liver.70 Undoubtedly, vectors that have been modified for a reduction in CpG motifs will have significant advantages for many forms of gene therapy, where the activation of innate immunity is not desirable. On the other hand, CpG motifs may be beneficial in the treatment of allergic diseases (see below), or in cancer gene therapy.

CpG ODNs as Immunotherapeutic Agents

CpG ODNs are finding increasing applications for immunotherapy (Table 1). The ability of ODNs carrying immununostimulatory CpG motifs (or ISS) to activate innate immune mechanisms has proven valuable in cancer immunotherapy.59,60 Importantly, the ODNs stimulate DCs and induce their maturation. These DCs are more effective at stimulating effector T cells and, furthermore, they secrete cytokines such as IL-6 which appear to protect effector cells against the suppressive effects of regulatory T cells.72 ISS can also stimulate NK cells and B cells, which contributes to anti-tumor immunity in some models.

Table 1. Examples of therapeutic applications of nucleic acid therapy with CpG or siRNA.

Table 1

Examples of therapeutic applications of nucleic acid therapy with CpG or siRNA.

CpGs have also been applied to the therapy of allergic diseases.73-81 The CpG-ODN can be coadministered with the allergen, or directly fused with that molecule. Notably, ovalbumin (OVA) conjugated to a CpG-ODN and administered intratracheally in mice was found to be 100-fold more effective at ameliorating OVA-induced asthma than a mixture of OVA and CpG-ODN.79,80 It is unclear why conjugation is more effective, but this may be related to increased uptake of the antigen by APCs, or colocalization of both molecules to the same APC. ODNs can alter Th1/Th2 balance in a favorable way (decreased Th2), possibly by stimulating production of Th1-type inflammatory cytokines such as IL-12 and IFNγ.79-81 In allergic diseases, at least three major modalities have produced positive therapeutic effects: (1) DNA vaccination against allergens; (2) immunization with allergen/ODN mixtures or allergen/ODN conjugates; and (3) administration of ODNs alone.

Several authors have documented that CpG-ODN therapy inhibits Th2 cytokine production, eosinophilic inflammation and airway hypersensitivity in murine models of asthma.76-81 The inhibition of eosinophilia is thought to be related to decreased IL-5 production (a Th2 cytokine). CpG ODNs were also found to be remarkably effective against allergic conjunctivitis. 75 Although most literature has focused on Th1/Th2 antagonism, the role of Th1 cells can be questioned, because CpG ODNs were protective against airway hypersensitivity in mice lacking IL-12 and IFNγ82 Indeed, some ODNs have been reported to induce IL-10, and protection against allergy might depend on the activity of IL-10-producing regulatory T cells, or other types of regulatory cells.82 In a murine model of chronic asthma, CpG ODNs also increased the amount of TGF-β1 in bronchoalveolar lavage fluid, possibly due to the action of Tr cells.78 However, it remains unclear which features of ODNs would make them suitable for the induction of regulatory cytokines, rather than inflammatory cytokines.

Detrimental Effects of Plasmid DNA or CpG ISS Motifs

It is of some concern that transfected muscle cells may be attacked and injured by the immune system following DNA vaccination against foreign antigens, and indeed this has been reported.83 A related concern is the production of pathogenic anti-DNA antibodies, potentially induced by plasmid DNA and its ISS motifs, but the risk appears relatively small. Indeed, B cells have mechanisms which prevent autoantibody production in response to CpG stimulation,84 although this tolerance can be broken.85 In lupus-prone mice, anti-dsDNA antibodies titers are increased by DNA vaccination. Surprisingly, lupus-like disease was either not altered or reduced in some studies.86,87 However, recent reports indicate that stimulation through TLR9 induces progression of renal disease in MRL-lpr/lpr (Fas deficient)88 and NZB x NZWF189 lupus-prone mice. Evidently, special caution should be exercised in administering CpG-bearing plasmids to patients with autoimmune diseases.

Local injection of stimulatory ODNs can induce inflammation. For instance intra-articular injection of these ODNs induces a form of arthritis, characterized by joint swelling, synovial hyperplasia and leukocytic infiltration.90,91 Interestingly, this form of arthritis is reduced by prior systemic administration of suppressive ODNs.90,91

The Potent Inhibitory Effects of TGF-β1 and Its Use in Gene Therapy

The most potent anti-inflammatory cytokine is TGF-β1, though IL-4, IL-10, and IL-13 have some similar effects, particularly through their action on macrophages. There is a plethora of information in the literature on the immunobiology of TGF-β,6,92-102 and only major points are mentioned here. At least three TGF-β isoforms exist in mammals, but TGF-β1 is the principal type produced by cells of the immune system. It is secreted in a latent form where mature TGF-β1 is associated with a precursor peptide (latency associated peptide (LAP) and latent TGF-β1-binding protein (LTBP). The active form can be generated in vitro by acidification of this complex, and is probably released in vivo through the action of plasmin and other proteases in inflammatory or other sites, though the mechanism is not fully elucidated. TGF-β1 receptors are expressed by almost all cells and, interestingly, this cytokine also binds to several matrix components in tissue. It has fibrogenic and angiogenic effects that contribute to wound healing.92,102

TGF-β1 is produced by regulatory T cells (Tr), particularly those designated Th3 and Tr1 (reviewed in refs. 95-98, 103). Importantly, TGF-β has also emerged as an important differentiation factor for Tr cells.95-98,104-109 In addition, this cytokine is produced by macrophages and many other cell types in various tissues. It exerts diverse immunoinhibitory effects on B lymphocytes, CD4+ T lymphocytes (Th1 or Th2), CTLs, NK cells, lymphokine-activated killer (LAK) cells, and macrophages.6,95-97 In macrophages, TGF-β1 antagonizes the activities of IFNγ and TNFα, and inhibits inducible nitric oxide synthase (iNOS) activity. This cytokine also alters expression of E-selectin and other adhesion molecules, and interferes with the adhesion of neutrophils and lymphocytes to endothelial cells. The potent immunosuppressive effects of TGF-β1 are most clearly demonstrated in studies of knockout (KO) mice, which die rapidly from a multi-organ inflammatory syndrome.

The fibrogenic and immunosuppressive effects of TGF-β1 overproduction have been linked to several pathologic conditions, particularly pulmonary fibrosis, glomerulopathy, systemic sclerosis, and chronic graft-versus-host disease (GVHD).6,102,110 This cytokine promotes corneal opacification (increased extracellular matrix, angiogenesis, cell infiltration) after injury or transplantation. Moreover, it is produced by most tumours, where it is capable of blocking anti-tumor immunity.111 High production of TGF-β1 has also been noted in chronic infectious diseases, where it hampers the elimination of pathogens.112 On the other hand, injection of a plasmid encoding TGF-β1 into skin wounds improved healing in diabetic mice.113 This might be related to the stimulation of fibroblast proliferation and collagen deposition, as well as the promotion of angiogenesis.

Cytokine Gene Therapy of Lupus

Administration of TGF-β1 is protective in several inflammatory conditions. In rodents, microgram amounts of either active or latent protein are required to achieve immunosuppressive effects.114-116 The delivery of TGF-β1 by gene transfer has been examined by several authors in classical models of autoimmunity or inflammatory disease (Table 2). We have shown that, among other advantages, this route obviates the time consuming and expensive process of TGF-β1 purification. Intramuscular (i.m.) injection of naked plasmid DNA encoding latent TGF-β1 (pCMV-TGF-β1) increases circulating levels of this cytokine by several folds, suppresses delayed-type hypersensitivity (DTH) and protects against autoimmune lesions.1,3,117,118 In most cases, a gene encoding latent TGF-β1 has been employed, and it is evident that the cytokine is released into the circulation and activated in vivo, although the mechanism of activation has not been clearly established. We hypothesize that at least part of the circulating TGF-β1 is activated at sites of inflammation, through the action of macrophages or other lymphoid cells. Although administration of a modified active form of the TGF-β1 gene is feasible, the fact that virtually all cells have receptors102 makes it likely that most of the TGF-β1 molecules would never reach their intended target, and numerous adverse effects would likely occur.

Table 2. Examples of successful plasmid-based TGF- β1 gene therapy.

Table 2

Examples of successful plasmid-based TGF- β1 gene therapy.

Raz and colleagues117,118 showed that direct injections of cDNA expression plasmids encoding IL-2, IL-4, or latent TGF-β1 into mouse skeletal muscle induce biological effects characteristic of these cytokines. Mice injected intramuscularly with a vector encoding IL-2 had enhanced humoral and cellular immune responses to an exogenous antigen, transferrin, which was delivered at a separate site. These IL-2 effects were abolished by coadministration of a vector directing synthesis of TGF-β1. The TGF-β1 vector alone depressed the anti-transferrin antibody response and caused an 8-fold increase in plasma TGF-β1 activity. The TGF-β1 plasmid injection did not cause muscle infiltration with monocytes or neutrophils and there was no evidence for fibrotic changes. Monthly injections of TGF-β1 plasmid DNA in these mice between 6 and 26 weeks of age prolonged survival of 70% at 26 weeks compared with 40% in the control group, decreased anti-chromatin and rheumatoid factor antibodies and induced a 50% decrease in total IgG production. Renal function was improved with reduced BUN levels and kidney inflammation as estimated by a histology, and these beneficial effects occurred in the apparent absence of local or systemic side effects. In contrast, injection of IL-2 cDNA resulted in decreased survival to 20% at 26 weeks, enhanced total IgG synthesis and autoantibody production with a 4.5-fold increase in anti-chromatin antibodies.

However, not all investigators have found IL-2 to be detrimental in lupus. For example, Gutierrez-Ramos and colleagues119 observed a beneficial effect for IL-2 on the disease progression in MRL lpr/lpr mice using live vaccinia recombinant viruses expressing the human IL-2 gene, e.g., prolonged survival, decreased autoantibody and rheumatoid factor titres, marked attenuation of kidney interstitial infiltration and intraglomerular proliferation, as well as clearance of synovial mononuclear infiltrates. Additionally, such inoculation resulted in drastically reduced double-negative T cells, improved thymic differentiation and restored normal values of mature cells in peripheral lymphoid organs. A caveat is that immune responses to vaccinia antigens could have altered the immune system and contributed to this beneficial effect. Indeed, the use of strongly antigenic viral vectors is a serious limitation for immunological studies.

Huggins and colleagues120,121 also studied the effects of IL-2 and TGF-β gene therapy on the progress of autoimmune disease in MRL lpr/lpr mice, using a different approach. The mice were treated orally with a nonpathogenic strain of Salmonella typhimurium bearing the aroA-aroD- mutations and carrying the murine genes encoding IL-2 or TGF-β1. This results in the in vivo uptake of the bacteria by some cells (e.g., phagocytes), gene transfer, synthesis and slow release of the cytokines, although the intracellular mechanisms of gene transfer are not fully elucidated. These investigators reported that, contrary to expectation, TGF-β1 gene therapy failed to ameliorate disease and generally produced effects opposite to those of IL-2 therapy. IL-2 restored the deficient T-cell proliferative response to mitogen and suppressed the autoantibody response and glomerulonephritis.

These conflicting results demonstrate the risks involved in using cytokines as therapeutic molecules. Most cytokines have complex pleiotropic actions, and may have stimulatory or inhibitory effects depending on their concentration, target tissue or cell, as well as interacting cytokines in the extra-cellular milieu. Indeed, the coactivity of multiple cytokines produced in inflammatory sites may produce effects that have never been documented in vitro. As a result, cytokines that are generally thought of as anti-inflammatory, such as TGF-β1, sometimes have inflammatory effects.6,122 Similarly, inflammatory cytokines, such as IL-12, are sometimes paradoxically protective. For example Hagiwara and colleagues123 found that administering a DNA plasmid encoding IL-12 to MRL-lpr/lpr mice significantly inhibited lymphadenopathy and splenomegaly. A significant decrease in serum IgG anti-DNA autoantibody titres was observed, and plasmid IL-12 therapy was also associated with a reduction in the proteinuria and glomerulonephritis. Serum IFNγ level was increased by inoculating the IL-12 encoding plasmid, suggesting that the cytokine balance was skewed towards a Th1-type response. This is surprising, since other authors found that neutralizing IFNγ is protective in this disease (see below). It may be that this plasmid also induced the production of regulatory cytokines that counterbalanced inflammatory cytokines, but we can only speculate on the mechanism at this point.

Recent studies also show that IFNγ can induce the expression of PD-1 ligand 1 (PD-L1) on DCs, endothelial cells, and other cells.124 This ligand binds to the inhibitory molecule PD-1 and turns off T-cell and B-cell responses. Other inhibitory receptors such as B and T lymphocyte attenuator (BTLA) show many similarities.125 Therefore, inflammatory cytokines may paradoxically activate negative regulatory mechanisms which protect against autoimmunity.

Applicability of TGF-β1 Gene Therapy to Various Inflammatory Diseases

In addition to lupus, administration of TGF-β1 is protective in several inflammatory conditions (Table 2). Kitani et al126 showed that a single intranasal dose of a plasmid encoding active TGF-β1 in mice prevented the development of Th1-dependent colitis induced by the haptenating reagent, 2,4,6-trinitrobenzene sulfonic acid (TNBS). Plasmid administration abrogated TNBS colitis after it had been established, and it led to the expression of TGF-β1 mRNA in the intestinal lamina propria, as well as the appearance of TGF-β1-producing T cells and macrophages in these tissues. These cells caused marked suppression of IL-12 and IFNγ production and enhancement of IL-10 production. Thus, TGF-β1 gene therapy appears to augment the production of TGF-β1 by various cells, including regulatory T cells. Interestingly, this therapy was not associated with fibrosis.

In a similar model of induced rat colitis, i.m. injection of a TGF-β1 expression plasmid also ameliorated colonic inflammation and ulceration.127 On histological examination, 50% of TGF-β1-plasmid treated rats had minimal or no ulceration and a significant decrease in mucosal leukotriene C4 generation, whereas 83% of control plasmid-treated rats had a maximal damage score. Similarly, others128 administered a TGF-β1 plasmid to rats with streptococcal cell wall (SCW)-induced arthritis. Systemic delivery of TFG-β1 initiated by i.m. injection of a single dose of 300 μg of plasmid DNA encoding TGF-β1, but not vector DNA, profoundly suppressed the subsequent evolution of chronic erosive disease. However, it should be noted that, unlike systemic administration, the intra-articular delivery of TGF-β1 can induce osteoarthritis-like inflammation.122 This may be related to the chemotactic, angiogenic and fibrogenic properties of this cytokine.

TGF-β1 gene therapy might also be applicable to the prevention of transplant rejection. Indeed, direct injection of plasmids encoding this cytokine into cardiac muscle protected mice against allogeneic heart transplant rejection.129-131 Plasmid-induced immunosuppression was localized to the area of the graft because plasmid injected remote from the graft was not protective, and systemic immunity was not affected. Similarly, the perfusion of donor hearts with DNA-liposome carrying the TGF-β1 gene prolonged allograft survival in approximately two-thirds of recipients.132 This was associated with reduced Th1 responses and an inhibition of alloantibody isotype switching. Transgene expression persisted for at least 60 days; however, long-term transfected allografts exhibited exacerbated fibrosis and neointimal development.

Cytokine Gene Therapy of Organ-Specific Autoimmune Diseases

We found that the injection of a TGF-β1 plasmid (100 μg/muscle in 2-4 muscles every 2 weeks; into the tibialis anterior or rectus femoris) considerably reduced the incidence of diabetes in NOD mice.1 In the cyclophosphamide (CYP)-accelerated form of this disease, there was a four-fold reduction in incidence. In nonCYP-treated mice (natural course), treatment reduced the incidence of diabetes by approximately 50 % over the course of several weeks, even when therapy was administered late in mice that already had insulitis. Semi-quantitative analysis of cytokine mRNA expression in the pancreas of treated mice revealed decreased levels of inflammatory cytokine mRNA. Detrimental effects were not noted and, in this respect, the use of latent TGF-β1 is probably an advantage since it can only act in sites where it can be activated, such as inflammatory lesions.

IL-4 and IL-10 have also been extensively studied as possible immunotherapeutic cytokines. Systemic administration of IL-4 or IL-10 protein to NOD mice, or transgenic expression of the IL-4 (but not IL-10) gene in their islets, prevents insulitis and diabetes.19,20,133,134 In accordance with this, IL-4 or IL-10 gene therapies, which promote Th2 activity, ameliorate this disease (Table 3). IL-10 is discussed separately in another section below. We compared the effects of delivering IL-4 with an IL-4/IgG1-Fc fusion gene, or inhibiting IFNγ with an IFNγR/IgG1-Fc fusion gene (this vector is described below).4 The positive effects of the soluble FNγ receptor were significant but less than IL-4/IgG1-Fc, and are described separately. Following i.m. delivery of the IL-4/IgG1-Fc vector serum levels were low (< 10 pg/ml), compared to other vectors we have tested. Nevertheless, IL-4/IgG1-Fc was potently active in vivo, consistent with other observations that this cytokine is immunomodulatory even at very low circulating levels.135 As in the case of TGF-β1gene therapy, NOD mice injected with the IL-4/IgG1-Fc plasmid were protected from inflammatory pancreatic-islet lesions and had a much lower incidence of diabetes.4 Other authors have reported similar protective effects of the IL-4 gene after epidermal gene-gun delivery of a plasmid136 or systemic adenoviral injections.137 Interestingly, protein administration in NOD mice must be started as early as 2 weeks of age to prevent diabetes, although we found that gene transfer of IL-4 was effective when started later.

Table 3. Examples of plasmid-based IL-4 or IL-10 gene therapy.

Table 3

Examples of plasmid-based IL-4 or IL-10 gene therapy.

We also examined the effects of TGF-β1 and IL-4/IgG1-Fc cDNA transfer in murine EAE.3 I.m. TGF-β1 plasmid delivery had pronounced downregulatory effects on T cell proliferation and production of IFNγ and TNFα, on in vitro restimulation with MBP. IL-4/IgG1-Fc vector administration also suppressed these responses, although much less than TGF-β1, and enhanced secretion of endogenous IL-4. Therapy resulted in a significant decrease in the severity of histopathologic inflammatory lesions. In the CNS, treatment with either vector suppressed IL-12 and IFNγ mRNA expression, while IL-4 and TGF-β1 mRNA levels were increased compared with control mice. Thus, cytokine plasmid treatment appeared to inhibit MBP-specific pathogenic Thl responses, while enhancing endogenous secretion of protective cytokines. We demonstrated that gene therapy with these vectors is an effective therapeutic strategy for EAE.

Croxford and coworkers138 recently reported that i.m. injection of plasmids encoding TGF-β1 and IL-4 failed to influence the clinical course of EAE. This discrepancy with our results may be due to the fact that no detectable plasmid-derived cytokine production was observed in their experiments. Furthermore, they administered plasmid DNA as a single dose in tibialis anterior muscles concurrently with MBP immunization. In our study, plasmid DNA was administered 48 hours before both the initial MBP priming and recall immunizations. Furthermore, we used a plasmid vector, VR1255, selected for high expression in skeletal muscle, and clearly superior for this purpose compared to most other vectors described in the literature. Nevertheless, Croxford et al138 observed that EAE was ameliorated by a single injection of therapeutic cytokine (IL-4, IFNβ, or TGF-β) plasmid DNA-cationic liposome complex directly into the CNS. DNA coding for a dimeric form of human p75 TNF receptor also improved the disease. This clearly demonstrates that tissue-specific expression of immune mediators relevant to an autoimmune disease can be accomplished with plasmid vectors.

IL-10 Gene Therapy for the Treatment of Inflammatory Diseases

IL-10 has many immunosuppressive and anti-inflammatory effects that could potentially block the autoimmune process at multiple steps.139-146 Indeed, IL-10 reduces MHC and B7.1/ B7.2 expression on APCs, induces T-cell anergy, promotes the differentiation of regulatory T cells (Tr1 type, see below), can increase T-cell apoptosis and can potently suppress pro-inflammatory cytokine expression/function, most notably IFNγ and TNF-α. It has suppressive effects on macrophages and DCs, inhibiting their maturation and consequential ability to promote CD4+ and CD8+ T cell responses. Thus, augmenting IL-10 production in vivo for the purpose of attenuating inflammatory responses and possibly inducing tolerance to autoantigens has been an area of intense scientific investigation.

In recent years, several gene therapy approaches have been developed for IL-10 therapy of inflammatory disorders (Table 3). For instance, systemic delivery of IL-10 by i.m. injection of a plasmid vector prevented autoimmune diabetes in NOD mice.147 In this case, IL-10 was detectable by ELISA in sera for more than two weeks after injection, and the incidence of diabetes was markedly curtailed. Interestingly, IL-10 did not prevent insulitis, and it appeared that the main effect of therapy was to skew the differentiation of T cells towards the Th2 pathway, consequently inhibiting diabetogenic Th1 cells. In light of more recent studies, however, we hypothesize that this cytokine also acted by inducing Tr1 differentiation. In contrast to this study, the intravenous injection of an IL-10 plasmid complexed to a degradable polymeric carrier, poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA), did ameliorate insulitis.148 Codelivery of IL-4 and IL-10 cDNA by this method (plasmid/PAGA carrier) also protected against insulitis and diabetes, and proved therapeutically superior to injection of either gene alone.149,150 In multiple low-dose streptozotocin (STZ)-induced diabetes (MDSD), i.m. delivery of an IL-10 plasmid also protected against insulitis and diabetes.151 Serum IFNγ levels were reduced, consistent with the pathogenic role of this cytokine in this form of diabetes.

In rat or mouse experimental autoimmune myocarditis, i.m. IL-10 or vIL-10-IgG-Fc gene transfer were both found to be protective.152-154 For instance, Lewis rats immunized with pig myosin to induce myocarditis were treated with an IL-10 plasmid, transferred into the tibialis anterior muscles by electroporation.152 With repeated DNA injections, the serum IL-10 levels were increased to > 250 pg/ml, and treated animal had significantly reduced myocardial inflammatory lesions and prolonged survival. Other investigators showed that injection of a plasmid carrying the fusion of vIL-10 with an immunoglobulin Fc fragment resulted in much higher circulating levels of protein (up to 195 ng/ml), which protected against mouse viral myocarditis.154

In a murine experimental autoimmune thyroiditis model, direct injection of an IL-10 plasmid into the thyroid gland considerably reduced the lymphocytic infiltration.155 Similarly, other authors156 delivered an IL-10 plasmid complexed to a mixture of liposomes and poly-L-Lysine to enhance transfection. There was a significant diminution in the proliferative T-cell response to thyroglobulin (the target antigen) and lower production of IFNγ or Th2 bias in the response. These studies demonstrate that nonviral IL-10 gene therapy can be applied in a tissue-localized fashion.

Inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis are chronic inflammatory disorders of the gastrointestinal tract where initiation and aggravation of the inflammatory process seem to be due to a massive local mucosal immune response where IL-10 correlates with protection.157,158 Multiple strategies of IL-10 treatment for IBD have been described and include systemic administration of recombinant IL-10, the use of genetically modified bacteria, gelatine microspheres containing IL-10, adenoviral vectors encoding IL-10 and IL-10-secreting regulatory T cells (see below).157 Although most results of recombinant IL-10 therapies are disappointing in clinical testing because of low efficacy or side effects, therapeutic strategies utilizing gene therapy may enhance mucosal delivery and increase the therapeutic response. Novel IL-10-related cytokines, including IL-19, IL-20, IL-22, IL-24, IL-26, IL-28 and IL-29, are involved in regulation of inflammatory immune responses. Thus, the use of IL-10 and IL-10-related cytokines may provide new insights into future cell-based and gene-based treatments against chronic inflammatory diseases like IBD.

Cytokine Gene Therapy and the Induction of Regulatory T Cells (Tr)

The use of cytokine gene therapy for the treatment and/or establishment of tolerance in chronic inflammatory conditions is expectedly associated with a state of reduced immunoreactivity to self-tissues. Investigators very often infer the existence of an induced/expanded regulatory T cell population underlying this state of hyporesposiveness, although regulation of T cell responses can readily occur in the functional absence of regulatory T cells. Indeed, the negative regulation of innate and adaptive immunity is highly complex, occurring at multiple levels through many disparate mechanisms.159 Nonetheless, the potential of inducing and selectively engaging specific peripheral immunoregulatory networks by means of innovative gene therapy strategies truly represents a novel and critical area of immunotherapy.

What are regulatory T cells and what is their therapeutic benefit? In an attempt to fine-tune and diversify its ability to control adaptive immune responses in a timely and efficient manner, the immune system has evolved numerous mechanisms, including Tr cells, to modulate and down-regulate immune responses at various locations and in various immune settings (fig.1).160,161 To this end, a network of Tr exists to assure T cell immunoregulation at multiple levels depending on the inflammatory burden and anatomical location. Indeed, a large number of Tr cell populations have been described, and for the most part, their definition has been based on their phenotype and, their relative cytokine production capabilities. In general, most of the described Tr cells arise after deliberate antigen exposure and include regulatory Th2 cells (which suppress Th1 associated responses), Th1 cells (which suppress Th2-cell-mediated responses), IL-10-producing Tr1 cells (a subset of IL-10-dependent, antigen-specific regulatory T cells), TGF-β1-secreting Th3 cells, CD8+, natural killer T (NKT), and γδ T cells.

Figure 1. Differentiation pathways of Tr cells.

Figure 1

Differentiation pathways of Tr cells. Natural and induced (adaptive) Tr cells arise through different pathways. While the natural Tr cells appear to differentiate in the thymus, the induced Tr cells develop in response to antigenic stimulation and are (more...)

More recently, naturally-occuring CD4+CD25+ Tr cells, which exist in the unperturbed T cell repertoire and do not arise from experimental immunostimulation, have emerged as a dominant T cell population capable of mediating peripheral tolerance to autoantigens, but whose functions have now been extended to include the regulation of T cell responses directed to foreign antigens. A seminal observation made by Sakaguchi and coworkers,162 and subsequent work,163,164 demonstrated that adoptive transfer of CD4+CD25+-depleted T cells induced several organ-specific autoimmune diseases in recipient, immunodeficient animals, including gastritis, diabetes, IBD, and thyroiditis. Furthermore, cotransfer of these cells with enriched CD4+CD25+ cells prevented autoimmunity, confirming the regulatory activity of the latter. These findings explained earlier studies, performed 4 decades earlier, which showed that day 3 thymectomy of select strains of neonatal mice disrupted Tr cell development and consequently lead to systemic autoimmunity. CD4+CD25+ Tr cells consitute 5-10% of CD4+ T cells in mice and humans, and their removal from peripheral immune systems also increases immunity to tumors and allografts. In addition to regulating self-responses, CD4+CD25+ Tr cells also control immunity to bacterial, fungal, protozoal, nematodal, and viral pathogens.

Several cytokines including IL-10 and TGF-β1, have been implicated in Tr effector functions, 103,141,165,166 and their relative contribution to the generation and effector function of Tr cells remains incompletely defined. While the requirement of cytokines via which Tr cells regulate T responses in vivo are largely determined by tissue- or context-dependent factors, the consensus view is that in vitro CD4+CD25+ Tr-mediated suppression adopts a contact-dependent, cytokine-independent mechanism of suppression.

The specific generational/stimulatory signals required for the selective expansion and survival in the periphery, particularly in autoimmune -prone subjects, is ill-defined. Failure to obtain compelling answers results, in part, from the fact that different experimental systems are often used, or largely from the fact that investigators routinely fail to discriminate naturally-occurring CD4+CD25+ T cells from CD25-expressing CD4+ T cell populations induced to acquire suppressor activity throughout the course of an immune response or as a result from a unique stimulatory condition. Thus, the bulk of current literature indicates that there are two general categories of CD4+CD25+ Tr cells (although there may be subtypes), which differ in their origin and effector mechanism. One Tr subset develops during the normal process of T cell maturation in the thymus, resulting in the generation of a naturally-occurring population of CD4+CD25+ Tr (nTr) cells that survives in the periphery poised to prevent potential autoimmune responses.160,161,164 The second subset of induced CD4+CD25+ Tr (iTr) cells whose precursor is thymically-derived, develops as a consequence of activation of classical naive T cell populations under particular conditions of antigen exposure, cytokine stimulation and/or costimulation.141,165,166 Thus, CD4+ T cells with regulatory function can be generated by the activation of mature, peripheral CD4+ T cells, and regulatory and pathogenic T cells can, in principle, be generated from the same mature T cell precursors, depending on qualitative and/or quantitative differences in antigen priming. Induced Tr cells can be generated in vivo or ex vivo from mature CD4+CD25- T cell populations under different stimulatory conditions including, antigen in the presence of immunosuppressive cytokines, such as IL-10 and TGF-β1, vitamin D3 and dexamethasone, CD40-CD40L blockade or immature DC populations. It must be noted that antigen exposure by intranasal, intradermal or oral route, seems to selectively induce the appearance of T cells with regulatory phenotype (Th3), whose function in vitro and in vivo generally occurs in a cytokine-dependent manner.103 Although distinct in nature, it is conceivable that peripheral T cell immunoregulation is assured by a functional synchrony between nTr cell and iTr cell subsets in order to control the activation and function of normal and autoimmune responses.

The relevance of IL-10 to Tr cell differentiation and function has received increasing attention in recent years.141,160,161,165-167 Although most investigators performing IL-10 gene therapy have not examined this question, a recent study provides interesting information. In accord with previous studies with other vectors, Goudy et al167 found that systemic treatment of NOD mice with high doses of recombinant adeno-associated virus (rAAV) vector expressing murine IL-10 reduced the severity of insulitis and completely prevented diabetes in all NOD mice, including older 12-week-old mice that are known to resist most forms of immunotherapy. Notably, IL-10 gene therapy dramatically increased the percentage of CD25-expressing CD4+ regulatory T cells, although the cellular origin and functional nature of these cells remains to be defined. Thus, IL-10 treatment of inflammatory conditions may potentially support, under certain circumstances, the induction of CD4+ Tr cells, which can control inflammation.

More recent evidence also suggests that T cell priming in the presence of TGF-β1 potently induces lymphocytes with regulatory potential, suggesting a possible alternate pathway for generating Tr cells in the periphery, as mentioned above.104-109 Thus, TGF-β1 was shown to induce Foxp3 expression and subsequent regulatory function in murine CD4+CD25- T cells in an IL-2-dependent fashion.108 Surprisingly, the induced Tr cells appeared to mediate their function in contact-dependent, and cytokine-independent fashion. In a related study, Peng et al167 showed that transient pulses of TGF-β1 in the islets during the priming phase of diabetes is sufficient to inhibit disease onset by promoting the cycling, expansion and activity of intra-islet CD4+CD25+ Tr cells which demonstrated features reminiscent of nTr cells: CD25, CTLA-4, and Foxp3 expression. Furthermore, adoptive transfer of these cells potently protected against diabetes. These findings indicate that TGF-β treatment may inhibit autoimmune diseases via the in situ induction, expansion and function of CD4+CD25+ Tr cells in vivo, thus providing a possible cellular mechanism by which TGF-β can promote immunosuppression and tolerance. Once again, the precise generational and functional relationship of these cells with naturally-occurring Tr cells remains to be defined.

Retroviral Transduction of T Cells and Adoptive Cell Therapy of Autoimmune Disease

Autoimmune disorders represent inappropriate immune responses directed at self-tissue. Antigen-specific CD4+ T cells and antigen-presenting dendritic cells (DCs) are important mediators in the pathogenesis of autoimmune disease and thus are ideal candidates for adoptive cellular gene therapy, an ex vivo approach to therapeutic gene transfer. As discussed in another chapter, retrovirally transduced primary T cells rapidly and preferentially home to the sites of inflammation in animal models of multiple sclerosis, arthritis, and diabetes. These cells, transduced with retroviral vectors to drive expression of various regulatory cytokines such as IL-4, IL-10, and IL-12p40 antagonists, deliver these immunoregulatory proteins to the inflamed lesions, providing therapy for autoimmune diseases.

Gene Therapy of Lupus with Cytokine Inhibitors

Cytokine inhibitors (usually antibodies or soluble cytokine receptors) are advantageously nontoxic and often long-lived in body fluids, compared with most cytokines, Most gene therapy studies of cytokine inhibitors have been carried out with viral vectors, and there is less experience with nonviral methods. However, we and others have shown that the plasmid-based transfer of cDNA encoding these molecules protects against several autoimmune diseases (Table 4). As mentioned above, we constructed an expression plasmid encoding an IFNγR/IgG1-Fc fusion protein.2,4,5 The appropriate murine cDNA segments were inserted into the plasmid VR1255 (Vical Inc., San Diego, CA), which is exceptionally effective in muscle.37 It has a CMV IE-EP, CMV intron A, and a rabbit beta-globin transcriptional terminator. COS-7 cells transfected with this plasmid secreted IFNγR/IgG1-Fc fusion protein in vitro as a disulfide-linked homodimer, with the expected biological activity.2 Thus, IFNγR/IgG1-Fc neutralized IFNγ-dependent NO production by macrophages (stimulated with IFNγ and lipopolysaccharide [LPS]). I.m. injections (100 μg naked DNA/muscle into 2 muscles, administered twice) of the IFNγR/IgG1-Fc plasmid in normal mice resulted in IFNγR/IgG1-Fc serum levels exceeding 100 ng/ml for months after treatment. Higher levels (> 200 ng/ml) were produced by repeated DNA injections.

Table 4. Examples of plasmid-based anticytokine gene therapy.

Table 4

Examples of plasmid-based anticytokine gene therapy.

Our studies showed that soluble receptor levels in the range of 100-200 ng/ml effectively blocked IFNγ-induced pathology in four different experimental models. The high-level and long-term expression of this vector, compared with many other plasmid vectors, may be related to the neutralization of IFNγ, since this cytokine can suppress transcription promoted by CMV IE-EP elements.

Many abnormalities in the cytokine network have been reported in lupus, but increased levels of IFNγ, as well as some IFNaβ species, in serum, lymphoid organs and inflamed tissues are prominent.18,168 In particular, the production of IFNγ is remarkably high in MRL-lpr/lpr lupus-prone mice.5,169 Therefore, it was of interest to determine if IFNγ could be blocked by a gene therapy approach. We inoculated an IFNγR/IgG1-Fc plasmid into lupus-prone and observed low level expression compared with a previous study in NOD and CD1 mice with the same vector. However, serum IFNγ levels of untreated MRL-lpr/lpr mice are very high, and it is possible the soluble receptor was removed after binding to IFNγ. Alternatively, residual IFNγ might have shut down the vector's IFNγ-sensitive CMV enhancer/promoter.

When in vivo electroporation was applied to enhance gene transfer in MRL lpr/lpr mice, serum IFNγR/IgG1-Fc levels, which had been < 10 ng/ml without electroporation, exceeded 100 ng/ml and, consequently, IFN-γ serum levels were markedly reduced.5 Thus, electroporation was remarkably effective, and it is likely that this technique will be even more relevant to other species. Indeed, as mentioned previously, in primates and other large mammals i.m. gene transfer of naked DNA is not as efficient as in rodents, but is greatly augmented by in vivo electroporation.

Treatment with the IFN-γR/IgG1 plasmid by i.m. injections, especially with electroporation, protected MRL lpr/lpr mice from early death, and reduced autoantibody titres, renal disease and histological markers of SLE-like disease.5 Most notably, when therapy was initiated in 4 month-old diseased mice, survival was extended beyond expectations, with 100% of the mice staying alive at 14 months of age compared with none in the control group. Remarkably, disease severity was reduced or even suppressed in the treated group.

The mechanism(s) by which IFNγ contributes to the pathogenesis of lupus is not clear, but there are important clues. IFNγ promotes production of autoantibodies of the IgG2a and IgG3 isotypes that efficiently activate complement. In addition, IgG3 has cryogenic properties. Furthermore, IFNγ enhances several pathogenic activities of macrophages and promotes inflammation in target tissues.

Other investigators have attempted to neutralize IFNγ in mouse lupus models using polyclonal and monoclonal antibodies (mAbs), as well as soluble IFNγR. These approaches, however, have limitations. For example, large quantities of mAbs would be required and may not achieve sufficient concentration in tissues to be effective, and/or they may be neutralized by the host immune response. With regard to soluble recombinant receptors, rapid turnover may affect efficacy and necessitates repeated administration. These constraints possibly explain previous negative results of anti-IFNγ mAb treatment of MRL lpr/lpr mice, and the finding that treatment with recombinant soluble IFNγR in NZBxNZWF1 lupus mice was effective only when initiated early, but not late, when IFNγ levels are significantly higher.170

The IFNγR/IgG1-Fc fusion protein produced in these studies is comprised of segments of endogenous murine proteins. Antibodies reactive with these proteins do not appear to be produced in treated mice, even after repeated injections of plasmid over several weeks. In this respect, it now clear that plasmids that do not encode immunogenic proteins, or plasmids injected into immunodeficient SCID mice, are expressed for longer periods. This may be related to the fact that myocytes encoding xenogeneic proteins can be attacked and killed by the immune system, as observed in DNA vaccination studies, and/or because locally produced IFNγ or other cytokines inhibit vector expression.

The addition of an Fc segment to a therapeutic protein is not always essential, but may confer significant advantages. The Fc portion simplifies purification of the recombinant protein by affinity chromatography, and the increase in size can prolong the half-life of small proteins in body fluids. For instance, the half-life of the truncated IFNγ receptor is quite short compared with a receptor/Fc fusion protein.171 Also, dimers are likely to have a higher avidity for their ligand, as is clearly the case with the IFNγ receptor.

Cytokine Inhibitors in Arthritis

IL-1Ra is an endogenous protein that can prevent the binding of IL-1 to its cell-surface receptors. IL-1Ra has shown promise in the therapy of arthritis, and is a candidate molecule for gene therapy. However, almost all studies have been conducted with viral vectors. Recently, Kim et al172 investigated i.m. plasmid-based IL-1ra therapy in the prevention of murine collagen-induced arthritis (CIA). In bovine type II collagen-immunized DBA/1 mice, delivery of IL-1ra cDNA significantly reduced joint pathology. Synovitis and cartilage erosion in knee joints were markedly reduced, and the expression of IL-1β was significantly decreased in the ankle joints of mice treated with IL-1Ra. This occurred despite the fact that the levels of IL-1Ra in sera and joints after i.m. injection of IL-1Ra DNA were significantly lower than when protein had been used in previous reports.

Kim and colleagues,173 Bloquel et al174 and Gould et al175 have also reported on the effectiveness of plasmid-based transfer of soluble TNF-receptor cDNA in CIA, as described in another chapter. As expected, in vivo electroporation increases the effectiveness of these vectors. In one study, the inhibition of established CIA was performed with a doxycycline regulated plasmid.175 Protection against CIA has also been achieved by transfer of IL-4176 and IL-10.177,178 These studies demonstrate that nonviral gene therapy can be effective against arthritis, at least when gene transfer is enhanced by electroporation.

Cytokine Inhibitors in Other Autoimmune Diseases

The transfer of cDNA encoding cytokine inhibitors protects against several autoimmune diseases (Table 4). IL-12 and IFNγ are usually detrimental in autoimmune diseases and, consequently, their neutralization is likely to be protective. These two cytokines are functionally related, since IL-12 induces IFNγ production by T cells and NK cells, while IFNγ mediates or augments many of the effects initiated by IL-12. The neutralization of IFNγ with mAbs or soluble receptors prevents NOD-mouse diabetes,2,8,9,179 as well as diabetes induced by administration of multiple low-dose STZ (MDSD) in other strains.171 CYP greatly accelerates disease in NOD mice, and the CYP- and STZ-induced diseases are both associated with a burst of systemic and intra-islet IFNγ release.180 Indeed, we observed that i.m. administration of an IFNγ expression plasmid accelerated disease in NOD mice,1 and others found that nondiabetes-prone transgenic mice expressing IFNγ in their islets developed insulitis/diabetes associated with a loss of tolerance to islet antigens.181 IFNγ, IL-12, IL-18 and other inflammatory cytokines are produced locally in the inflamed islets of NOD mice.19,20 Furthermore, microarray analysis of the islets of CYP-treated mice revealed that IFNγ dominated the changes in gene expression to a striking degree.182 Surprisingly, gene expression related to Tr cells was not markedly altered. IFNγ is toxic to islet cells, particularly in combination with IL-1 and TNFa (reviewed in refs. 19, 20). Also, it could act by activating macrophages, stimulating Th1 cells, or augmenting CTL and NK activity. All these cells have the potential to injure or kill islet cells.

In vivo, administration of our IFNγR/IgG1-Fc vector almost completely blocked the systemic IFNγ activity induced by either STZ (CD-1 or C57BL/6 mice) or CYP (NOD mice).2 Moreover, this plasmid was protective in either natural or drug-induced models of autoimmune diabetes,2,4 in agreement with the postulated pathogenic role of IFNγ. In each case, therapy reduced the severity insulitis and the frequency of diabetes which is secondary to this lesion. It should be noted, however, that this anti-cytokine therapy was more effective in the induced models of diabetes (STZ of CYP), presumably because IFNγ plays a more important role in the pathogenesis of these diseases. Nevertheless, IFNγR/IgG1-Fc gene therapy protected NOD mice and, interestingly, this was superior to IFNγ gene knockout which has only a modest effect.183 The reason is unclear, but mice deficient in IFNγ from fetal life may develop compensatory mechanisms.

RNA Interference with Nonviral Vectors

It is now well established that double-stranded RNA (dsRNA) induces gene silencing in a sequence specific way.184-187 Double-stranded siRNAs (short inhibitory RNAs) are produced from dsRNA through the activity of an RNase III family endonuclease denoted Dicer.184-187 They are generally 21 to 23 nucleotide long and have overhanging ends consisting of two nucleotides. siRNAs interact with a large multi-component enzyme termed RNA-induced silencing complex (RISC), to bind a fully complementary mRNA sequence, which results in endonucleolytic cleavage of the mRNA. RISC has two key components, i.e., siRNAs and Argonaute family proteins, and it is the Argonaute2 protein that actually cuts the mRNA.188 The cleaved RNA is further degraded by cellular exonuclease activities. siRNAs provide a stronger method of gene silencing than either antisense molecules or ribozymes. They are short enough to avoid induction of an interferon response, and this increases their research applicability and therapeutic potential immensely. Indeed, several in vivo applications have already been reported (Table 1).

Other types of short RNAs have equally impressive gene silencing properties.187,189 Diverse eukaryotic species, including humans, possess noncoding regulatory endogenous hairpin RNAs (microRNAs [miRNA]), that can interact with RISC and silence genes. However, unlike siRNAs, miRNAs do not induce degradation of mRNA, but rather partially bind to its 3' UTR and block translation.187,189 Thus, RISC can interfere with protein synthesis and this is the dominant mechanisms used by miRNAs in mammals. In addition, short RNAs can shut down gene expression by inducing specific methylation of promoters, in several species including humans.190

It is reasonably simple to deliver siRNAs to cells in vitro, with methods such as cationic lipids or electroporation, but delivery in vivo is more difficult. For in vivo administration, hydrodynamic delivery and electroporation have both been employed. For example, McCaffrey et al191 and Lewis et al192 silenced genes in vivo in mice by injecting siRNA in the tail vein under pressure. Liver uptake of siRNA was observed, and a sequence-specific gene silencing effect in that organ persisted for 3 or 4 days. Other studies showed that the intravenous injection of Fas-specific siRNA protected against hepatitis and hepatic necrosis induced by administration of either concanavalin A (Con A) or anti-Fas monoclonal antibodies.193 Caspase 8 siRNA also protected against acute liver failure in similar models.194 Remarkably, improved survival due to caspase 8 RNA interference was observed when treatment was applied during ongoing acute liver failure. A limitation of these methods is that the siRNA is distributed to multiple organs.

Hagstrom et al195 demonstrated delivery of plasmid DNA or siRNA by injection into the distal veins of limbs transiently isolated from the circulation by a tourniquet. Delivery to myocytes was facilitated by the rapid injection of sufficient volume to permit extravasation of the nucleic acid solution into muscle tissue. With this method, they reported siRNA-mediated gene silencing in rat and primate limb muscle. Kishida et al196 delivered siRNA duplexes corresponding to reporter genes by electroporation into the tibial muscle of mice expressing these reporter genes (transgenic or vector induced). As little as 0.05 μg of siRNA almost completely blocked the expression of a reporter gene from 10 μg of plasmid DNA, for at least one week. In transgenic mice, green fluorescent protein expression was also effectively blocked in cells receiving the complementary siRNA.

Some disadvantages of these methods include the high cost of producing sufficient quantities of siRNA, transient in vivo activity and, in some cases, distribution of the siRNA to tissues outside the target area. These limitations can be circumvented by the administration of siRNA plasmid or viral (adenoviral, retroviral or lentiviral) vectors.185-187,197,198 Viral vectors, however, are limited by the biological effects they produce, and nonviral methods are often preferable. Furthermore, nonviral methods can be adapted for both systemic and tissue-specific delivery. For example, target tissues have included tumors or limb muscle. Most of these vectors advantageously employ Pol III promoters such as U6, tRNA or H1, although Pol II constructs are feasible.187 Various designs are possible,187 e.g., vectors producing two separate complementary RNA strands, or producing short hairpin RNAs (shRNAs). The shRNAs are processed in vivo by Dicer, to generate active siRNAs. The vector can also produce a modified miRNA that is also processed by Dicer. The use of plasmid or viral vectors allows the introduction of tissue-specific or drug-sensitive promoters, to either limit expression to a target tissue or limit expression to a desired period of time.

The applications of siRNA technology are numerous. It represents a powerful research tool for studying physiological and pathological gene function.184-187 The in vitro or in vivo delivery of siRNA of obvious interest for investigative or therapeutic purposes in infectious and inflammatory diseases, as well as cancer.186,187,191-198 Indeed, its therapeutic potential has been clearly demonstrated in murine models of viral hepatitis, where both synthetic and plasmid-based siRNA therapy has been effective in suppressing the expression of viruses.199 Electroporation-enhanced plasmid siRNA delivery, with appropriate modifications, should be applicable to many tissues.

Nonviral Gene Transfer in Humans

There have been questions as to whether nonviral gene therapy and/or DNA vaccination are effective in large mammals. However, plasmid-based gene transfer for DNA vaccination or other purposes has been successfully performed in pigs, dogs, ruminants, horses, nonhuman primates and humans.200,201 Therapeutic levels of angiogenic factors have been generated in human skeletal and cardiac muscle.202,203 Of note, in the future, gene transfer could be greatly improved by introducing electroporation, hydrodynamic delivery or other new approaches. Nonviral gene transfer is particularly applicable to cancer therapy. For instance, some authors have investigated gene therapy for malignant gliomas by in vivo transduction with the human IFNβ gene using cationic liposomes,208 and other clinical trials are ongoing.

Most of the human studies have been in the area of DNA vaccination. Notably, immune responses can be generated against malaria antigens by i.m. DNA vaccination, and recent studies point to heterologous plasmid/virus prime-boost strategies as an effective method of generating immunity.204-207 Antigen-reactive T cells are readily induced, but antibody responses are usually of low magnitude. Vaccination is well tolerated, with either few or no side effects. In fact, nonviral DNA transfer into humans has had a remarkable safety profile, and is attracting more attention for this reason. Therefore, there is no obvious contra-indication for the use of these techniques in patients with autoimmune disease.

Conclusions and Future Prospects

The gene therapy of autoimmune diseases holds great promise. Unlike protein therapy, it allows long-term and relatively constant delivery of many cytokines or cytokine inhibitors at therapeutic levels even after one or a few treatments. Moreover, organ-specific delivery of mediators is feasible, either by direct injection of vectors into tissues or, as discussed in other chapters, by ex vivo transduction of cells, which can be reimplanted. Autoreactive T cells can also be transduced ex vivo, and transferred into recipients where they home to target tissues.

Viral and nonviral vectors have been used to protect against organ-specific and systemic autoimmune diseases in several models. TGF-β1 gene therapy protects against murine lupus, autoimmune diabetes, EAE, arthritis and colitis. IL-4 and IL-10 have also proved effective in several autoimmune conditions.

In our laboratories, we relied on administration of plasmid DNA into skeletal muscle. These vectors are nonimmunogenic and can be expressed in muscle for months. Naked DNA injection usually generates relatively low levels of circulating cytokines, which can often be advantageous, since these mediators are active at very low levels, while high levels can be severely toxic. When higher levels of expression are desired, as with cytokine inhibitors, this can be accomplished by applying in vivo electroporation.

The delivery of inhibitory soluble cytokine receptors, or other cytokine inhibitors, has significant advantages over other methods. The neutralization of IFNγ and IL-1 is particularly effective in models of lupus and arthritis, respectively. These receptors have no direct toxic or adverse effects other than depressed immunity, but only as related to the neutralization of one cytokine. Most, if not all, immunosuppressive drugs have many adverse effects and much broader suppressive activity. Cytokines can be blocked with monoclonal antibodies, but even humanized immunoglobulins can give rise to a neutralizing immune response in the recipient. In contrast, soluble receptors made only of self elements are much less likely to be neutralized.

The use of nonviral nucleic acids in experimental therapy is constantly expanding. This includes the application of CpG ODNs to the immunotherapy of cancer and allergic diseases. The most remarkable new development, however, is the introduction of siRNA-based therapeutic agents. Indeed, synthetic or vector-delivered siRNAs are powerful new tools for gene silencing, and their potential therapeutic applications are numerous. However, targeting the in vivo delivery of these molecules to a specific tissue is difficult, and nonviral methods of nucleic acid transfer, such as electroporation or hydronamic delivery, have advantages in terms of simplicity, effectiveness and safety.

Many tools are now available to the immunologist, at least experimentally, to treat inflammatory diseases, but few are as promising as the gene therapy or other nucleic acid transfer approaches.

Acknowledgements

Our studies were funded by the Juvenile Diabetes Research Foundation, the Canadian Diabetes Association, the National Cancer Institute of Canada, the Canadian Institutes of Health Research, the St. Michael's Hospital Foundation, and the National Institutes of Health (USA; grants AR31203, AG15061, and AR39555). We thank Vical Inc. (San Diego, California) for providing the VR1255 expression plasmid used in our studies.

References

1.
Piccirillo CA, Chang Y, Prud'homme GJ. Transforming growth factor beta-1 (TGF-β1) somatic gene therapy prevents autoimmune disease in NOD mice. J Immunol. 1998;161:3950–3956. [PubMed: 9780163]
2.
Prud'homme GJ, Chang Y. Prevention of autoimmune diabetes by intramuscular gene therapy with a nonviral vector encoding an interferon-gamma receptor/IgG1 fusion protein. Gene Ther. 1999;6:771–777. [PubMed: 10505100]
3.
Piccirillo CA, Prud'homme GJ. Prevention of experimental allergic encephalomyelitis by intramuscular gene transfer with cytokine-encoding plasmid vectors. Hum Gene Ther. 1999;10:1915–1922. [PubMed: 10466625]
4.
Chang Y, Prud'homme GJ. Intramuscular administration of expression plasmids encoding interferon-gamma-receptor/IgG1 or IL-4/IgG1 chimeric proteins protects from autoimmunity. J Gene Med. 1999;1:415–423. [PubMed: 10753067]
5.
Lawson BR, Prud'homme GJ, Chang Y. et al. Treatment of mouse lupus with cDNA encoding IFN-γR/Fc. J Clin Invest. 2000;106:207–215. [PMC free article: PMC314313] [PubMed: 10903336]
6.
Prud'homme GJ, Piccirillo CA. Inhibitory effects of transforming growth factor beta-1 in autoimmune diseases. J Autoimmun. 2000;14:23–42. [PubMed: 10648114]
7.
Prud'homme GJ. Gene therapy of autoimmune diseases with vectors encoding regulatory cytokines or inflammatory cytokine inhibitors. J Gene Med. 2000;2:222–232. [PubMed: 10953913]
8.
Prud'homme GJ, Lawson BR, Chang Y. et al. Immunotherapeutic gene transfer into muscle. Trends Immunol. 2001;22:149–155. [PubMed: 11286730]
9.
Prud'homme GJ, Lawson BR, Theofilopoulos AN. Anticytokine gene therapy of autoimmune diseases. Expert Opin Biol Ther. 2001;1:359–373. [PubMed: 11727511]
10.
Piccirillo CA, Prud'homme GJ. Immune modulation by plasmid DNA-mediated cytokine gene transfer. Curr Pharm Des. 2003;9:83–94. [PubMed: 12570678]
11.
Piccirillo CA, Prud'homme GJ. Gene therapy with plasmids encoding cytokine- or cytokine receptor-IgG chimeric proteins. Methods Mol Biol. 2003;215:153–70. [PubMed: 12512296]
12.
Mageed RA, Prud'homme GJ. Immunopathology and gene therapy of lupus. Gene Ther. 2003;10:861–874. [PubMed: 12732872]
13.
Dinarello CA. The IL-1 family and inflammatory diseases. Clin Exp Rheumatol. 2002;20(5 Suppl 27):S1–13. [PubMed: 14989423]
14.
Feldmann M, Brennan FM, Williams RO. et al. The transfer of a laboratory based hypothesis to a clinically useful therapy: The development of anti-TNF therapy of rheumatoid arthritis. Best Pract Res Clin Rheumatol. 2004;18:59–80. [PubMed: 15123038]
15.
Vilcek J, Feldmann M. Historical review: Cytokines as therapeutics and targets of therapeutics. Trends Pharmacol Sci. 2004;25:201–209. [PubMed: 15063084]
16.
Feldmann M, Brennan FM, Foxwell BM. et al. The role of TNF alpha and IL-1 in rheumatoid arthritis. Curr Dir Autoimmun. 2001;3:188–199. [PubMed: 11791466]
17.
Feldmann M, Maini RN. Anti-TNF alpha therapy of rheumatoid arthritis: What have we learned? Annu Rev Immunol. 2001;19:163–916. [PubMed: 11244034]
18.
Theofilopoulos AN, Lawson BR. Tumour necrosis factor and other cytokines in murine lupus. Ann Rheum Dis. 1999;58(Suppl 1):I49–155. [PMC free article: PMC1766580] [PubMed: 10577973]
19.
Rabinovitch A, Suarez-Pinzon WL. Role of cytokines in the pathogenesis of autoimmune diabetes mellitus. Rev Endocr Metab Disord. 2003;4:291–299. [PubMed: 14501180]
20.
Rabinovitch A. Immunoregulation by cytokines in autoimmune diabetes. Adv Exp Med Biol. 2003;520:159–193. [PubMed: 12613578]
21.
Vandenbroeck K, Alloza I, Gadina M. et al. Inhibiting cytokines of the interleukin-12 family: Recent advances and novel challenges. J Pharm Pharmacol. 2004;56:145–160. [PubMed: 15005873]
22.
Segal BM. Experimental autoimmune encephalomyelitis: Cytokines, effector T cells, and antigen-presenting cells in a prototypical Th1-mediated autoimmune disease. Curr Allergy Asthma Rep. 2003;3:86–93. [PubMed: 12543000]
23.
Lin MT, Pulkkinen L, Uitto J. et al. The gene gun: current applications in cutaneous gene therapy. Int J Dermatol. 2000;39:161–170. [PubMed: 10759952]
24.
Furth PA. Gene transfer by biolistic process. Mol Biotechnol. 1997;7:139–143. [PubMed: 9219228]
25.
El-Aneed A. An overview of current delivery systems in cancer gene therapy. J Control Release. 2004;94:1–14. [PubMed: 14684267]
26.
McMahon JM, Wells DJ. Electroporation for gene transfer to skeletal muscles: Current status. Bio Drugs. 2004;18:155–165. [PubMed: 15161333]
27.
Herweijer H, Wolff JA. Progress and prospects: Naked DNA gene transfer and therapy. Gene Ther. 2003;10:453–458. [PubMed: 12621449]
28.
Bigey P, Bureau MF, Scherman D. In vivo plasmid DNA electrotransfer. Curr Opin Biotechnol. 2002;13:443–447. [PubMed: 12459335]
29.
Zhang G, Budker V, Wolff JA. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum Gene Ther. 1999;10:1735–1737. [PubMed: 10428218]
30.
Zhang G, Budker V, Williams P. et al. Efficient expression of naked dna delivered intraarterially to limb muscles of nonhuman primates. Hum Gene Ther. 2001;12:427–438. [PubMed: 11242534]
31.
Hagstrom JE, Hegge J, Zhang G. et al. A facile nonviral method for delivering genes and siRNAs to skeletal muscle of mammalian limbs. Mol Ther. 2004;10:386–398. [PubMed: 15294185]
32.
Miller DL, Pislaru SV, Greenleaf JE. Sonoporation: Mechanical DNA delivery by ultrasonic cavitation. Somat Cell Mol Genet. 2002;27:115–134. [PubMed: 12774945]
33.
Hosseinkhani H, Aoyama T, Ogawa O. et al. Ultrasound enhances the transfection of plasmid DNA by nonviral vectors. Curr Pharm Biotechnol. 2003;4:109–122. [PubMed: 12678886]
34.
Wells DJ. Gene therapy progress and prospects: Electroporation and other physical methods. Gene Ther. 2004;11:1363–1369. [PubMed: 15295618]
35.
Wolff JA, Malone RW, Williams P. et al. Direct gene transfer into mouse muscle in vivo. Science. 1990;247:1465–1468. [PubMed: 1690918]
36.
Wolff JA, Williams P, Acsadi G. et al. Conditions affecting direct gene transfer into rodent muscle in vivo. Biotechniques. 1991;11:474–485. [PubMed: 1793583]
37.
Hartikka J, Sawdey M, Cornefert-Jensen F. et al. An improved plasmid DNA expression vector for direct injection into skeletal muscle. Hum Gene Ther. 1996;7:1205–1217. [PubMed: 8793545]
38.
Song K, Chang Y, Prud'homme GJ. Regulation of T-helper-1 versus T-helper-2 activity and enhancement of tumor immunity by combined DNA-based vaccination and nonviral cytokine gene transfer. Gene Ther. 2000;7:481–492. [PubMed: 10757021]
39.
Dupuis M, Denis-Mize K, Woo C. et al. Distribution of DNA vaccines determines their immunogenicity after intramuscular injection in mice. J Immunol. 2000;165:2850–2858. [PubMed: 10946318]
40.
Muramatsu T, Nakamura A, Park HM. In vivo electroporation: a powerful and convenient means of nonviral gene transfer to tissues of living animals. Int J Mol Med. 1998;1:55–62. [PubMed: 9852198]
41.
Mir LM, Bureau MF, Gehl J. et al. High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc Natl Acad Sci USA. 1999;96:4262–4267. [PMC free article: PMC16320] [PubMed: 10200250]
42.
Lucas Ml, Heller R. Immunomodulation by electrically enhanced delivery of plasmid DNA encoding IL-12 to murine skeletal muscle. Mol Ther. 2001;3:47–53. [PubMed: 11162310]
43.
Mathiesen I. Electropermeabilization of skeletal muscle enhances gene transfer in vivo. Gene Ther. 1999;6:508–514. [PubMed: 10476210]
44.
Rizzuto G, Cappelletti M, Maione D. et al. Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation. Proc Natl Acad Sci USA. 1999;96:6417–6422. [PMC free article: PMC26896] [PubMed: 10339602]
45.
Harrison RL, Byrne BJ, Tung L. Electroporation-mediated gene transfer in cardiac tissue. FEBS Letters. 1998;435:1–5. [PubMed: 9755847]
46.
Somiari S, Glasspool-Malone J, Drabick JJ. et al. Theory and in vivo application of electroporative gene delivery. Mol Ther. 2000;2:178–187. [PubMed: 10985947]
47.
Bettan M, Emmanuel F, Darteil R. et al. High level protein secretion into blood circulation after electric pulse-mediated gene transfer into skeletal muscle. Mol Ther. 2000;2:204–210. [PubMed: 10985950]
48.
Martin JB, Young JL, Benoit Jn. et al. Gene transfer to intact mesenteric arteries by electroporation. J Vasc Res. 2000;37:372–380. [PMC free article: PMC4152911] [PubMed: 11025400]
49.
Dev SB, Rabussay DP, Widera G. et al. Medical applications of electroporation. IEEE Transactions on Plasma Science. 2000;28:206–223.
50.
Suzuki T, Shin BC, Fujikura K. et al. Direct gene transfer into rat liver cells by in vivo electroporation. FEBS Letters. 1998;425:436–440. [PubMed: 9563509]
51.
Nishi T, Yoshizato K, Yamashiro S. et al. High-efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation. Cancer Res. 1996;56:1050–1055. [PubMed: 8640760]
52.
Neumann E, Kakorin S, Toensing K. Fundamentals of electroporative delivery of drugs and genes. Bioelectrochem & Bioenergetics. 1999;48:3–16. [PubMed: 10228565]
53.
Singh BN, Dwivedi C. Antitumor drug delivery by tissue electroporation. Anti-Cancer Drugs. 1999;10:139–146. [PubMed: 10211543]
54.
Zhou ZF, Peretz Y, Chang Y. et al. Intramuscular gene transfer of soluble B7.1/IgG(1) fusion cDNA induces potent antitumor immunity as an adjuvant for DNA vaccination. Cancer Gene Ther. 2003;10(6):491–9. [PubMed: 12768195]
55.
Gothelf A, Mir LM, Gehl J. Electrochemotherapy: Results of cancer treatment using enhanced delivery of bleomycin by electroporation. Cancer Treat Rev. 2003;29:371–387. [PubMed: 12972356]
56.
Mir LM. Therapeutic perspectives of in vivo cell electropermeabilization. Bioelectrochemistry. 2001;53:1–10. [PubMed: 11206915]
57.
Heller R, Gilbert R, Jaroszeski MJ. Clinical applications of electrochemotherapy. Adv Drug Deliv Rev. 1999;35:119–129. [PubMed: 10837693]
58.
Kishida T, Asada H, Itokawa Y. et al. Electrochemo-gene therapy of cancer: Intratumoral delivery of interleukin-12 gene and bleomycin synergistically induced therapeutic immunity and suppressed subcutaneous and metastatic melanomas in mice. Mol Ther. 2003;8:738–745. [PubMed: 14599806]
59.
Klinman DM. Immunotherapeutic uses of CpG oligodeoxynucleotides. Nat Rev Immunol. 2004;4:249–258. [PubMed: 15057783]
60.
Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol. 2002;20:709–760. [PubMed: 11861616]
61.
Vollmer J, Weeratna R, Payette P. et al. Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities. Eur J Immunol. 2004;34:251–62. [PubMed: 14971051]
62.
Rothenfusser S, Tuma E, Endres S. et al. Plasmacytoid dendritic cells: The key to CpG. Hum Immunol. 2002;63:1111–1119. [PubMed: 12480254]
63.
Hochrein H, O'Keeffe M, Wagner H. Human and mouse plasmacytoid dendritic cells. Hum Immunol. 2002;63:1103–1110. [PubMed: 12480253]
64.
Ishii KJ, Gursel I, Gursel M. et al. Immunotherapeutic utility of stimulatory and suppressive oligodeoxynucleotides. Curr Opin Mol Ther. 2004;6:166–174. [PubMed: 15195929]
65.
Klinman DM, Zeuner R, Yamada H. et al. Regulation of CpG-induced immune activation by suppressive oligodeoxynucleotides. Ann N Y Acad Sci. 2003;1002:112–123. [PubMed: 14751829]
66.
Qin L, Ding Y, Pahud DR. et al. Promoter attenuation in gene therapy: Interferon-gamma and tumor necrosis factor-alpha inhibit transgene expression. Hum Gene Ther. 1997;8:2019–2029. [PubMed: 9414251]
67.
Chen D, Murphy B, Sung R. et al. Adaptive and innate immune responses to gene transfer vectors: Role of cytokines and chemokines in vector function. Gene Ther. 2003;10:991–998. [PubMed: 12756420]
68.
Bromberg JS, Debruyne LA, Qin L. Interactions between the immune system and gene therapy vectors: Bidirectional regulation of response and expression. Adv Immunol. 1998;69:353–409. [PubMed: 9646848]
69.
Reyes-Sandoval A, Ertl HC. CpG methylation of a plasmid vector results in extended transgene product expression by circumventing induction of immune responses. Mol Ther. 2004;9:249–261. [PubMed: 14759809]
70.
Chen ZY, He CY, Ehrhardt A. et al. Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo. Mol Ther. 2003;8:495–500. [PubMed: 12946323]
71.
Darquet AM, Rangara R, Kreiss P. et al. Minicircle: An improved DNA molecule for in vitro and in vivo gene transfer. Gene Ther. 1999;6:209–218. [PubMed: 10435105]
72.
Pasare C, Medzhitov R. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science. 2003;299:1033–1036. [PubMed: 12532024]
73.
Horner AA, Raz E. DNA-based immunotherapeutics for allergic disease In: Raz E, ed.Microbial DNA and Host Immunity Humana Press,2002279–299.
74.
Kitigaki K, Kline JN. CpG oligodeoxynucleotides in asthma In: Raz E, ed.Microbial DNA and Host Immunity Humana Press,2002301–314.
75.
Keane-Myers A, Chan CC. Modulation of allergic conjunctivitis by immunostimulatory DNA sequence oligonucleotides In: Raz E, ed.Microbial DNA and Host Immunity Humana Press,2002315–325.
76.
Jain VV, Kline JN. CpG DNA: Immunomodulation and remodelling of the asthmatic airway. Expert Opin Biol Ther. 2004;4:1533–1540. [PubMed: 15335319]
77.
Jain VV, Kitagaki K, Kline JN. CpG DNA and immunotherapy of allergic airway diseases. Clin Exp Allergy. 2003;33:1330–1335. [PubMed: 14519136]
78.
Jain VV, Kitagaki K, Businga T. et al. CpG-oligodeoxynucleotides inhibit airway remodeling in a murine model of chronic asthma. J Allergy Clin Immunol. 2002;110:867–872. [PubMed: 12464952]
79.
Shirota H, Sano K, Kikuchi T. et al. Regulation of murine airway eosinophilia and Th2 cells by antigen-conjugated CpG oligodeoxynucleotides as a novel antigen-specific immunomodulator. J Immunol. 2000;164:5575–5582. [PubMed: 10820231]
80.
Datta SK, Cho HJ, Takabayashi K. et al. Antigen-immunostimulatory oligonucleotide conjugates: Mechanisms and applications. Immunol Rev. 2004;199:217–226. [PubMed: 15233737]
81.
Datta SK, Takabayashi K, Raz E. The therapeutic potential of antigen-oligonucleotide conjugates. Ann NY Acad Sci. 2003;1002:105–111. [PubMed: 14751828]
82.
Kitagaki K, Jain VV, Businga TR. et al. Immunomodulatory effects of CpG oligodeoxynucleotides on established th2 responses. Clin Diagn Lab Immunol. 2002;9:1260–1269. [PMC free article: PMC130087] [PubMed: 12414759]
83.
Davis HL, Millan CL, Watkins SC. Immune-mediated destruction of transfected muscle fibers after direct gene transfer with antigen-expressing plasmid DNA. Gene Ther. 1997;4:181–188. [PubMed: 9135731]
84.
Rui L, Vinuesa CG, Blasioli J. et al. Resistance to CpG DNA-induced autoimmunity through tolerogenic B cell antigen receptor ERK signaling. Nat Immunol. 2003;4:594–600. [PubMed: 12740574]
85.
Tran TT, Reich 3rdCF, Alam M. et al. Specificity and immunochemical properties of anti-DNA antibodies induced in normal mice by immunization with mammalian DNA with a CpG oligonucleotide as adjuvant. Clin Immunol. 2003;109:278–287. [PubMed: 14697742]
86.
Pizetsky DS. The antigenic properties of bacterial DNA in normal and aberrant immunity. Springer Semin Immunopathol. 2000;22:153–166. [PubMed: 10944810]
87.
Gilkeson GS, Ruiz P, Pippen AM. et al. Modulation of renal disease in autoimmune NZB/NZW mice by immunization with bacterial DNA. J Exp Med. 1996;183:1389–1397. [PMC free article: PMC2192478] [PubMed: 8666897]
88.
Anders HJ, Vielhauer V, Eis V. et al. Activation of toll-like receptor-9 induces progression of renal disease in MRL-Fas(lpr) mice. FASEB J. 2004;18:534–536. [PubMed: 14734643]
89.
Hasegawa K, Hayashi T. et al. Synthetic CpG oligodeoxynucleotides accelerate the development of lupus nephritis during preactive phase in NZB x NZWF1 mice. Lupus. 2003;12:838–845. [PubMed: 14667100]
90.
Zeuner RA, Verthelyi D, Gursel M. et al. Influence of stimulatory and suppressive DNA motifs on host susceptibility to inflammatory arthritis. Arthritis Rheum. 2003;48:1701–1707. [PubMed: 12794839]
91.
Zeuner RA, Ishii KJ, Lizak MJ. et al. Reduction of CpG-induced arthritis by suppressive oligodeoxynucleotides. Arthritis Rheum. 2002;46:2219–2224. [PubMed: 12209528]
92.
Schiller M, Javelaud D, Mauviel A. TGF-beta-induced SMAD signaling and gene regulation: Consequences for extracellular matrix remodeling and wound healing. J Dermatol Sci. 2004;35:83–92. [PubMed: 15265520]
93.
ten DijkeP, Hill CS. New insights into TGF-beta-Smad signalling. Trends Biochem Sci. 2004;29:265–273. [PubMed: 15130563]
94.
Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature. 2003;425:577–584. [PubMed: 14534577]
95.
Wahl SM, Chen W. TGF-beta: How tolerant can it be? Immunol Res. 2003;28:167–179. [PubMed: 14713712]
96.
Wahl SM, Swisher J, McCartney-Francis N. et al. TGF-beta: The perpetrator of immune suppression by regulatory T cells and suicidal T cells. J Leukoc Biol. 2004;76:15–24. [PubMed: 14966194]
97.
Luethviksson BR, Gunnlaugsdottir B. Transforming growth factor-beta as a regulator of site-specific T-cell inflammatory response. Scand J Immunol. 2003;58:129–38. [PubMed: 12869133]
98.
Levings MK, Bacchetta R, Schulz U. et al. The role of IL-10 and TGF-beta in the differentiation and effector function of T regulatory cells. Int Arch Allergy Immunol. 2002;129:263–276. [PubMed: 12483031]
99.
Leask A, Abraham DJ. TGF-beta signaling and the fibrotic response. FASEB J. 2004;18:816–827. [PubMed: 15117886]
100.
Bommireddy R, Doetschman T. TGF-beta, T-cell tolerance and anti-CD3 therapy. Trends Mol Med. 2004;10:3–9. [PMC free article: PMC2796480] [PubMed: 14720578]
101.
Siegel PM, Massague J. Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer. Nat Rev Cancer. 2003;3:807–821. [PubMed: 14557817]
102.
Howe PH. Transforming growth factor beta In: Thomson AW, Lotze MT, eds.The Cytokine Handbook Fourth Edition Academic Press,20031119–1152.
103.
Wu HY, Weiner HL. Oral tolerance. Immunol Res. 2003;28:265–284. [PubMed: 14713719]
104.
Fu S, Zhang N, Yopp AC. et al. TGF-beta Induces Foxp3 + T-Regulatory Cells from CD4 + CD25 - Precursors. Am J Transplant. 2004;4:1614–1627. [PubMed: 15367216]
105.
Schramm C, Huber S, Protschka M. et al. TGF-beta regulates the CD4+CD25+ T-cell pool and the expression of Foxp3 in vivo. Int Immunol. 2004;16:1241–1249. [PubMed: 15249539]
106.
Park HB, Paik DJ, Jang E. et al. Acquisition of anergic and suppressive activities in transforming growth factor-beta-costimulated CD4+CD25- T cells. Int Immunol. 2004;16:1203–1213. [PubMed: 15237111]
107.
Cobbold SP, Castejon R, Adams E. et al. Induction of foxP3+ regulatory T cells in the periphery of T cell receptor transgenic mice tolerized to transplants. J Immunol. 2004;172:6003–6010. [PubMed: 15128783]
108.
Zheng SG, Wang JH, Gray JD. et al. Natural and induced CD4+CD25+ cells educate CD4+CD25- cells to develop suppressive activity: The role of IL-2, TGF-beta, and IL-10. J Immunol. 2004;172:5213–5221. [PubMed: 15100259]
109.
Fantini MC, Becker C, Monteleone G. et al. Cutting edge: TGF-beta induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and down-regulation of Smad7. J Immunol. 2004;172:5149–5153. [PubMed: 15100250]
110.
Flanders KC. Smad3 as a mediator of the fibrotic response. Int J Exp Pathol. 2004;85:47–64. [PMC free article: PMC2517464] [PubMed: 15154911]
111.
Gorelik L, Flavell RA. Transforming growth factor-beta in T-cell biology. Nat Rev Immunol. 2002;2:46–53. [PubMed: 11905837]
112.
Reed SG. TGF-beta in infections and infectious diseases. Microbes Infect. 1999;1:1313–1325. [PubMed: 10611760]
113.
Chesnoy S, Lee PY, Huang L. Intradermal injection of transforming growth factor-beta1 gene enhances wound healing in genetically diabetic mice. Pharm Res. 2003;20:345–350. [PubMed: 12669952]
114.
Racke MK, Dhib-Jalbut S, Cannella B. et al. Prevention and treatment of chronic relapsing experimental allergic encephalomyelitis by transforming growth factor-beta 1. J Immunol. 1991;146:3012–3017. [PubMed: 1707929]
115.
Wallick SC, Figari IS, Morris RE. et al. Immunoregulatory role of transforming growth factor beta (TGF-beta) in development of killer cells: Comparison of active and latent TGF-beta 1. J Exp Med. 1990;172:1777–1784. [PMC free article: PMC2188774] [PubMed: 2258706]
116.
Kuruvilla AP, Shah R, Hochwald GM. et al. Protective effect of transforming growth factor beta 1 on experimental autoimmune diseases in mice. Proc Natl Acad Sci USA. 1991;88:2918–2921. [PMC free article: PMC51351] [PubMed: 2011600]
117.
Raz E, Watanabe A, Baird SM. et al. Systemic immunological effects of cytokine genes injected into skeletal muscle. Proc Natl Acad Sci. 1993;90:4523–4527. [PMC free article: PMC46544] [PubMed: 8506293]
118.
Raz E, Duddler J, Lotz M. et al. Modulation of disease activity in murine systemic lupus erythematosus by cytokine gene delivery. Lupus. 1995;4:286–292. [PubMed: 8528225]
119.
Gutierrez-Ramos JC, Andreu JL. et al. Recovery from autoimmunity of MRL/lpr mice after infection with an interleukin-2/vaccinia recombinant virus. Nature. 1990;346:271–274. [PubMed: 1973822]
120.
Huggins ML, Huang FP, Xu D. et al. Modulation of autoimmune disease in the MRL-lpr/lpr mouse by IL-2 and TGF-beta1 gene therapy using attenuated Salmonella typhimurium as gene carrier. Lupus. 1999;8:29–38. [PubMed: 10025597]
121.
Huggins ML, Huang FP, Xu D. et al. Modulation of the autoimmune response in lupus mice by oral administration of attenuated Salmonella typhimurium expressing the IL-2 and TGF-beta genes. Ann NY Acad Sci. 1997;815:499–502. [PubMed: 9186709]
122.
van BeuningenHM, Glansbeek HL, van der Kraan PM. et al. Osteoarthritis-like changes in the murine knee joint resulting from intra-articular transforming growth factor-beta injections. Osteoarthritis Cartilage. 2000;8:25–33. [PubMed: 10607496]
123.
Hagiwara E, Okubo T, Aoki I. et al. IL-12-encoding plasmid has a beneficial effect on spontaneous autoimmune disease in MRL/MP-lpr/lpr mice. Cytokine. 2000;12:1035–41. [PubMed: 10880249]
124.
Khoury SJ, Sayegh MH. The roles of the new negative T cell costimulatory pathways in regulating autoimmunity. Immunity. 2004;20:529–38. [PubMed: 15142522]
125.
Carreno BM, Collins M. BTLA: A new inhibitory receptor with a B7-like ligand. Trends Immunol. 2003;24:524–527. [PubMed: 14552835]
126.
Kitani A, Fuss IJ, Nakamura K. et al. Treatment of experimental (Trinitrobenzene sulfonic acid) colitis by intranasal administration of transforming growth factor (TGF)-beta1 plasmid: TGF-beta1-mediated suppression of T helper cell type 1 response occurs by interleukin (IL)-10 induction and IL-12 receptor beta2 chain downregulation. J Exp Med. 2000;192:41–52. [PMC free article: PMC1887715] [PubMed: 10880525]
127.
Giladi E, Raz E, Karmeli F. et al. Transforming growth factor-beta gene therapy ameliorates experimental colitis in rats. Eur J Gastroenterol Hepatol. 1995;7:341–347. [PubMed: 7600140]
128.
Song XY, Gu M, Jin WW. et al. Plasmid DNA encoding transforming growth factor-beta1 suppresses chronic disease in a streptococcal cell wall-induced arthritis model. J Clin Invest. 1998;101:2615–2621. [PMC free article: PMC508851] [PubMed: 9637694]
129.
Qin L, Chavin KD, Ding Y. et al. Gene transfer for transplantation. Prolongation of allograft survival with transforming growth factor-beta 1. Ann Surg. 1994;220:508–518. [PMC free article: PMC1234424] [PubMed: 7944661]
130.
Qin L, Chavin KD, Ding Y. et al. Multiple vectors effectively achieve gene transfer in a murine cardiac transplantation model. Immuno suppression with TGF-beta 1 or vIL-10. Transplantation. 1995;59:809–816. [PubMed: 7701573]
131.
Qin L, Ding Y, Bromberg JS. Gene transfer of transforming growth factor-beta 1 prolongs murine cardiac allograft survival by inhibiting cell-mediated immunity. Hum Gene Ther. 1996;7:1981–1988. [PubMed: 8930658]
132.
Chan SY, Goodman RE, Szmuszkovicz JR. et al. DNA-liposome versus adenoviral mediated gene transfer of transforming growth factor beta1 in vascularized cardiac allografts: Differential sensitivity of CD4+ and CD8+ T cells to transforming growth factor beta1. Transplantation. 2000;70:1292–1301. [PubMed: 11087143]
133.
Hill N, Sarvetnick N. Cytokines: Promoters and dampeners of autoimmunity. Curr Opin Immunol. 2002;14:791–797. [PubMed: 12413531]
134.
Gallichan WS, Balasa B, Davies JD. et al. Pancreatic IL-4 expression results in islet-reactive Th2 cells that inhibit diabetogenic lymphocytes in the nonobese diabetic mouse. J Immunol. 1999;163:1696–1703. [PubMed: 10415077]
135.
Ishii KJ, Weiss WR, Ichino M. et al. Activity and safety of DNA plasmids encoding IL-4 and IFN gamma. Gene Ther. 1999;6:237–244. [PubMed: 10435108]
136.
Cameron MJ, Strathdee CA, Holmes KD. et al. Biolistic-mediated interleukin 4 gene transfer prevents the onset of type 1 diabetes. Hum Gene Ther. 2000;11:1647–1656. [PubMed: 10954899]
137.
Cameron MJ, Arreaza GA, Waldhauser L. et al. Immunotherapy of spontaneous type 1 diabetes in nonobese diabetic mice by systemic interleukin-4 treatment employing adenovirus vector-mediated gene transfer. Gene Ther. 2000;7:1840–1846. [PubMed: 11110416]
138.
Croxford JL, Triantaphyllapoulos K, Podhajcer LL. et al. Cytokine gene therapy in experimental allergic encephalomyelitis by injection of plasmid DNA-cationic liposome complex into the central nervous system. J Immunol. 1998;160:5181–5187. [PubMed: 9590271]
139.
Moore KW, de Waal Malefyt R. et al. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol. 2001;19:683–765. [PubMed: 11244051]
140.
Weiss E, Mamelak AJ, La Morgia S. et al. The role of interleukin 10 in the pathogenesis and potential treatment of skin diseases. J Am Acad Dermatol. 2004;50:657–675. [PubMed: 15097948]
141.
Groux H, Cottrez F. The complex role of interleukin-10 in autoimmunity. J Autoimmun. 2003;20:281–285. [PubMed: 12791313]
142.
Roncarolo MG, Battaglia M, Gregori S. The role of interleukin 10 in the control of autoimmunity. J Autoimmun. 2003;20:269–272. [PubMed: 12791310]
143.
Bettelli E, Nicholson LB, Kuchroo VK. IL-10, a key effector regulatory cytokine in experimental autoimmune encephalomyelitis. J Autoimmun. 2003;20:265–267. [PubMed: 12791309]
144.
Asadullah K, Sterry W, Volk HD. Interleukin-10 therapy—review of a new approach. Pharmacol Rev. 2003;55:241–269. [PubMed: 12773629]
145.
Nitta Y, Tashiro F, Tokui M. et al. Systemic delivery of interleukin 10 by intramuscular injection of expression plasmid DNA prevents autoimmune diabetes in nonobese diabetic mice. Hum Gene Ther. 1998;9:1701–1707. [PubMed: 9721080]
146.
Koh JJ, Ko KS, Lee M. et al. Degradable polymeric carrier for the delivery of IL-10 plasmid DNA to prevent autoimmune insulitis of NOD mice. Gene Ther. 2000;7:2099–2104. [PubMed: 11223991]
147.
Ko KS, Lee M, Koh JJ. et al. Combined administration of plasmids encoding IL-4 and IL-10 prevents the development of autoimmune diabetes in nonobese diabetic mice. Mol Ther. 2001;4:313–316. [PubMed: 11592833]
148.
Lee M, Ko KS, Oh S. et al. Prevention of autoimmune insulitis by delivery of a chimeric plasmid encoding interleukin-4 and interleukin-10. J Control Release. 2003;88:333–342. [PubMed: 12628339]
149.
Zhang ZL, Shen SX, Lin B. et al. Intramuscular injection of interleukin-10 plasmid DNA prevented autoimmune diabetes in mice. Acta Pharmacol Sin. 2003;24:751–756. [PubMed: 12904273]
150.
Watanabe K, Nakazawa M, Fuse K. et al. Protection against autoimmune myocarditis by gene transfer of interleukin-10 by electroporation. Circulation. 2001;104:1098–1100. [PubMed: 11535562]
151.
Nakano A, Matsumori A, Kawamoto S. et al. Cytokine gene therapy for myocarditis by in vivo electroporation. Hum Gene Ther. 2001;12:1289–1297. [PubMed: 11440622]
152.
Adachi O, Nakano A, Sato O. et al. Gene transfer of Fc-fusion cytokine by in vivo electroporation: Application to gene therapy for viral myocarditis. Gene Ther. 2002;9:577–583. [PubMed: 11973633]
153.
Zhang ZL, Lin B, Yu LY. et al. Gene therapy of experimental autoimmune thyroiditis mice by in vivo administration of plasmid DNA coding for human interleukin-10. Acta Pharmacol Sin. 2003;24:885–890. [PubMed: 12956936]
154.
Batteux F, Trebeden H, Charreire J. Curative treatment of experimental autoimmune thyroiditis by in vivo administration of plasmid DNA coding for interleukin-10. Eur J Immunol. 1999;29:958–963. [PubMed: 10092100]
155.
Li MC, He SH. IL-10 and its related cytokines for treatment of inflammatory bowel disease. World J Gastroenterol. 2004;10:620–625. [PMC free article: PMC4716896] [PubMed: 14991925]
156.
Braat H, Peppelenbosch MP, Hommes DW. Interleukin-10-based therapy for inflammatory bowel disease. Expert Opin Biol Ther. 2003;3:725–731. [PubMed: 12880373]
157.
Prud'homme GJ. Altering immune tolerance therapeutically: The power of negative thinking. J Leukoc Biol. 2004;75:586–599. [PubMed: 14657212]
158.
Piccirillo CA, Thornton AM. Cornerstone of peripheral tolerance: Naturally occurring CD4+CD25+ regulatory T cells. Trends Immunol. 2004;25:374–380. [PubMed: 15207505]
159.
Piccirillo CA, Shevach EM. Naturally-occurring CD4+CD25+ immunoregulatory T cells: Central players in the arena of peripheral tolerance. Semin Immunol. 2004;16:81–88. [PubMed: 15036231]
160.
Sakaguchi S, Sakaguchi N, Asano M. et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol. 1995;155:1151–1164. [PubMed: 7636184]
161.
Asano M, Toda M, Sakaguchi N. et al. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J Exp Med. 1996;184:387–396. [PMC free article: PMC2192701] [PubMed: 8760792]
162.
Sakaguchi S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol. 2004;22:531–562. [PubMed: 15032588]
163.
Cottrez F, Groux H. Specialization in tolerance: Innate CD(4+)CD(25+) versus acquired TR1 and TH3 regulatory T cells. Transplantation. 2004;77(1 Suppl):S12–5. [PubMed: 14726762]
164.
Powrie F, Read S, Mottet C. et al. Control of immune pathology by regulatory T cells. Novartis Found Symp. 2003;252:92–98. [PubMed: 14609214]
165.
Peng Y, Laouar Y, Li MO. et al. TGF-beta regulates in vivo expansion of Foxp3-expressing CD4+CD25+ regulatory T cells responsible for protection against diabetes. Proc Natl Acad Sci USA. 2004;101:4572–4577. [PMC free article: PMC384788] [PubMed: 15070759]
166.
Santiago-Raber ML, Baccala R, Haraldsson KM. et al. Type-I interferon receptor deficiency reduces lupus-like disease in NZB mice. J Exp Med. 2003;197:777–788. [PMC free article: PMC2193854] [PubMed: 12642605]
167.
Prud'homme GJ, Kono DH, Theofilopoulos AN. Quantitative polymerase chain reaction analysis reveals marked overexpression of interleukin-1 beta, interleukin-10 and interferon-gamma mRNA in the lymph nodes of lupus-prone mice. Mol Immunol. 1995;32:495–503. [PubMed: 7783752]
168.
Ozmen L, Roman D, Fountoulakis M. et al. Experimental therapy of systemic lupus erythematosus: The treatment of NZB/W mice with mouse soluble interferon-gamma receptor inhibits the onset of glomerulonephritis. Eur J Immunol. 1995;25:6–12. [PubMed: 7843255]
169.
Kurschner C, Ozmen L, Garotta G. et al. IFN-gamma receptor-Ig fusion proteins. Half-life, immunogenicity, and in vivo activity. J Immunol. 1992;149:4096–4100. [PubMed: 1460292]
170.
Kim JM, Jeong JG, Ho SH. et al. Protection against collagen-induced arthritis by intramuscular gene therapy with an expression plasmid for the interleukin-1 receptor antagonist. Gene Ther. 2003;10:1543–1550. [PubMed: 12907945]
171.
Kim JM, Ho SH, Hahn W. et al. Electro-gene therapy of collagen-induced arthritis by using an expression plasmid for the soluble p75 tumor necrosis factor receptor-Fc fusion protein. Gene Ther. 2003;10:1216–1224. [PubMed: 12858186]
172.
Bloquel C, Bessis N, Boissier MC. et al. Gene therapy of collagen-induced arthritis by electrotransfer of human tumor necrosis factor-alpha soluble receptor I variants. Hum Gene Ther. 2004;15:189–201. [PubMed: 14975191]
173.
Gould DJ, Bright C, Chernajovsky Y. Inhibition of established collagen-induced arthritis with a tumour necrosis factor-alpha inhibitor expressed from a self-contained doxycycline regulated plasmid. Arthritis Res Ther. 2004;6:R103–113. [PMC free article: PMC400428] [PubMed: 15059273]
174.
Kageyama Y, Koide Y, Uchijima M. et al. Plasmid encoding interleukin-4 in the amelioration of murine collagen-induced arthritis. Arthritis Rheum. 2004;50:968–975. [PubMed: 15022341]
175.
Saidenberg-Kermanac'h N, Bessis N. et al. Efficacy of interleukin-10 gene electrotransfer into skeletal muscle in mice with collagen-induced arthritis. J Gene Med. 2003;5:164–171. [PubMed: 12539154]
176.
Miyata M, Sasajima T, Sato H. et al. Suppression of collagen induced arthritis in mice utilizing plasmid DNA encoding interleukin 10. J Rheumatol. 2000;27:1601–1605. [PubMed: 10914840]
177.
Campbell IL, Kay TW, Oxbrow L. et al. Essential role for interferon-gamma and interleukin-6 in autoimmune insulin-dependent diabetes in NOD/WEHI mice. J Clin Invest. 1991;87:739–742. [PMC free article: PMC296368] [PubMed: 1899431]
178.
Cockfield SM, Ramassar V, Urmson J. et al. Multiple low dose streptozotocin induces systemic MHC expression in mice by triggering T cells to release IFN-gamma. J Immunol. 1989;142:1120–1128. [PubMed: 2521659]
179.
Sarvetnick N, Shizuru J, Liggitt D. et al. Loss of pancreatic islet tolerance induced by beta-cell expression of interferon-gamma. Nature. 1990;346:844–847. [PubMed: 2118234]
180.
Matos M, Park R, Mathis D. et al. Progression to islet destruction in a cyclophosphamide-induced transgenic model: A microarray overview. Diabetes. 2004;53:2310–2321. [PubMed: 15331540]
181.
Hultgren B, Huang X, Dybdal N. et al. Genetic absence of gamma-interferon delays but does not prevent diabetes in NOD mice. Diabetes. 1996;45:812–817. [PubMed: 8635658]
182.
Jones SW, Souza PM, Lindsay MA. siRNA for gene silencing: a route to drug target discovery. Curr Opin Pharmacol. 2004;4:522–527. [PubMed: 15351359]
183.
Caplen NJ. Gene therapy progress and prospects. Downregulating gene expression: The impact of RNA interference. Gene Ther. 2004;11:1241–1248. [PubMed: 15292914]
184.
Ichim TE, Li M, Qian H. et al. RNA interference: A potent tool for gene-specific therapeutics. Am J Transplant. 2004;4:1227–1236. [PMC free article: PMC7175948] [PubMed: 15268723]
185.
Wadhwa R, Kaul SC, Miyagishi M. et al. Know-how of RNA interference and its applications in research and therapy. Mutat Res. 2004;567:71–84. [PubMed: 15341903]
186.
Liu J, Carmell MA, Rivas FV. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science. 2004;305:1437–1441. [PubMed: 15284456]
187.
Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–297. [PubMed: 14744438]
188.
Kawasaki H, Taira K. Induction of DNA methylation and gene silencing by short interfering RNAs in human cells. Nature. 2004;431:211–217. [PubMed: 15311210]
189.
McCaffrey AP, Meuse L, Pham TT. et al. RNA interference in adult mice. Nature. 2002;418:38–39. [PubMed: 12097900]
190.
Lewis DL, Hagstrom JE, Loomis AG. et al. Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nat Genet. 2002;32:107–108. [PubMed: 12145662]
191.
Song E, Lee SK, Wang J. et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med. 2003;9:347–351. [PubMed: 12579197]
192.
Zender L, Hutker S, Liedtke C. et al. Caspase 8 small interfering RNA prevents acute liver failure in mice. Proc Natl Acad Sci USA. 2003;100(13):7797–7802. [PMC free article: PMC164667] [PubMed: 12810955]
193.
Hagstrom JE, Hegge J, Zhang G. et al. A facile nonviral method for delivering genes and siRNAs to skeletal muscle of mammalian limbs. Mol Ther. 2004;10:386–398. [PubMed: 15294185]
194.
Kishida T, Asada H, Gojo S. et al. Sequence-specific gene silencing in murine muscle induced by electroporation-mediated transfer of short interfering RNA. J Gene Med. 2004;6:105–110. [PubMed: 14716682]
195.
Devroe E, Silver PA. Therapeutic potential of retroviral RNAi vectors. Expert Opin Biol Ther. 2004;4:319–327. [PubMed: 15006726]
196.
Morris KV, Rossi JJ. Anti-HIV-1 gene expressing lentiviral vectors as an adjunctive therapy for HIV-1 infection. Curr HIV Res. 2004;2:185–191. [PubMed: 15078182]
197.
Radhakrishnan SK, Layden TJ, Gartel AL. RNA interference as a new strategy against viral hepatitis. Virology. 2004;323:173–181. [PubMed: 15193913]
198.
Manoj S, Babiuk LA, van Drunen Littel-van den Hurk S. Approaches to enhance the efficacy of DNA vaccines. Crit Rev Clin Lab Sci. 2004;41:1–39. [PubMed: 15077722]
199.
Prud'homme GJ. DNA vaccination against tumors J Gene Med 2004. in press .
200.
Isner JM. Myocardial gene therapy. Nature. 2002;415:234–239. [PubMed: 11805848]
201.
Freedman SB, Vale P, Kalka C. et al. Plasma vascular endothelial growth factor (VEGF) levels after intramuscular and intramyocardial gene transfer of VEGF-1 plasmid DNA. Hum Gene Ther. 2002;13:1595–1603. [PubMed: 12228014]
202.
Moore AC, Hill AV. Progress in DNA-based heterologous prime-boost immunization strategies for malaria. Immunol Rev. 2004;199:126–143. [PubMed: 15233731]
203.
Moorthy VS, Imoukhuede EB, Keating S. et al. Phase 1 evaluation of 3 highly immunogenic prime-boost regimens, including a 12-month reboosting vaccination, for malaria vaccination in Gambian men. Infect Dis. 2004;189:2213–2219. [PubMed: 15181568]
204.
Epstein JE, Charoenvit Y, Kester KE. et al. Safety, tolerability, and antibody responses in humans after sequential immunization with a PfCSP DNA vaccine followed by the recombinant protein vaccine RTS,S/AS02A. Vaccine. 2004;22:1592–1603. [PubMed: 15068840]
205.
McConkey SJ, Reece WH, Moorthy VS. et al. Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans. Nat Med. 2003;9:729–735. [PubMed: 12766765]
206.
Yoshida J, Mizuno M, Fujii M. et al. Human gene therapy for malignant gliomas (glioblastoma multiforme and anaplastic astrocytoma) by in vivo transduction with human interferon beta gene using cationic liposomes. Hum Gene Ther. 2004;15:77–86. [PubMed: 14965379]
Copyright © 2000-2013, Landes Bioscience.
Bookshelf ID: NBK6462