Uveitis: Mechanisms and recent advances in therapy

Clinica Chimica Acta 411 (2010) 1165–1171

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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m

Invited critical review

Uveitis: Mechanisms and recent advances in therapy Arpna Srivastava, Medha Rajappa, Jasbir Kaur ⁎ Department of Ocular Biochemistry, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi 110 029, India

a r t i c l e

i n f o

Article history: Received 17 February 2010 Received in revised form 15 April 2010 Accepted 15 April 2010 Available online 21 April 2010 Keywords: Uveitis Intraocular inflammation Cytokine Gene therapy

a b s t r a c t Uveitis is a sight threatening inflammatory disorder that affects all ages and remains a significant cause of visual loss. Animal models of autoimmune and inflammatory disease in the eye allow the scientist and clinician to study the basic mechanism of the disease, and serve as templates for the development of therapeutic approaches. The accumulating knowledge of the various steps that are involved in the pathogenesis make it possible to devise specific strategies that disrupt discrete stages in the process. Some of the strategies are in the process of being translated to the clinic. New technologies emerge, promising more specific and more easily applied therapies. In this review, we will concentrate specifically on mechanisms and recent advances in the therapy of uveitis. © 2010 Elsevier B.V. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Immune mechanisms in uveitis . . . . . . . . . . . . . . . . . . . . . 3. Immunomodulation . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Various therapeutic approaches for uveitis . . . . . . . . . . . . . . . 5. Chemokines and their role in immunotherapy for intraocular inflammation 6. Gene therapy and its role in uveitis . . . . . . . . . . . . . . . . . . 7. Gene therapy using immunoglobulins . . . . . . . . . . . . . . . . . 8. RNA interference (RNAi) as therapeutic approach in uveitis . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Uveitis is the most common form of intraocular inflammation and remains a significant cause of visual loss. Current treatment for uveitis employs systemic medications that have severe side effects and are globally immunosuppressive. Clinically, chronic progressive or relapsing forms of non-infectious uveitis are treated with topical and/or systemic corticosteroids. In addition to steroids, macrolides such as cyclosporine and rapamycin are used, and in some cases cytotoxic agents such as cyclophosphamide and chlorambucil, and antimetabolites such as azathioprine, methotrexate, and lefunomide. While often effective, these drugs have potential serious side effects and they compromise protective immunity to pathogens [1]. A more specific approach to therapy that will target primarily the cells involved in

⁎ Corresponding author. Tel.: + 91 11 26593161; fax: + 91 11 26588919. E-mail address: [email protected] (J. Kaur). 0009-8981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2010.04.017

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pathogenesis and leave the rest of the immune system alone is urgently needed. Thus, there is urgent need to develop effective immunotherapeutic strategies that are non-toxic and that specially target the pathogenic cell population. The increased knowledge in immunology and the progresses of pharmacology have improved our treatment of autoimmune diseases. The main anti-inflammatory effects of corticosteroids are an attenuation of the hypersensibility reactions, a sequestration of intravascular lymphocytes and an inhibition of the production of cytokines and eicosanoids. The non-steroidal anti-inflammatory drugs (NSAIDs) form another group of medications particularly useful for the treatment of chronic uveitis. Several cyclooxigenase-2 inhibitory medications are at the moment under clinical investigation and some are commercially available. One of their characteristics is to present less of the most undesirable side effects seen with conventional NSAIDs like irritation of the gastrointestinal tract and platelets aggregation inhibition. Agents like cyclophosphamide, leukeran, imuran, methotrexate and cyclosporin have been used extensively

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for the treatment of severe uveitis. Because of its efficacy and safety, methotrexate is the best immunosuppressive agent to be tried for the treatment of chronic uveitis. However, immunosuppressive treatments and corticosteroids have many side effects and are not very selective. To improve our therapeutic arsenal, other treatments are being investigated for the treatment of severe uveitis. Significant advances in the diagnosis and therapy for uveitis have been made to improve the quality of care for patients with ocular inflammatory diseases. While traditional ophthalmic examination techniques, fluorescein angiography, and optical coherence tomography continue to play a major role in the evaluation of patients with uveitis, the advent of spectral domain optical coherence tomography and fundus autofluorescence into clinical practice provides additional information about disease processes. Polymerase chain reaction and cytokine diagnostics have also continued to play a greater role in the evaluation of patients with inflammatory diseases. Their ophthalmic indications have continued to expand, improving the therapeutic armentarium of specialists treating uveitis.

the notion that it is causally involved in human disease. The clear advantage of the antigen specific approach is that it targets only the cells specific to disease, and leaves the rest of the immune system unaffected. However, in many autoimmune entities the target antigens are not known, or the process of epitope spreading and antigen spreading may have occurred, and more than one antigen becomes involved. The alternative approach, still more specific than a general immunosuppressant, is to target activated lymphocytes based on common markers or processes involved in immune activation and/or function. One example of this would be therapy directed at activation receptors such as the IL-2 receptor, based on the premise that they will be expressed by the pathogenic effector T cells driving the disease. Although, at least in this particular case, the mechanisms involved appear to be much more complex than was initially assumed, the immunotherapeutic paradigm of targeting activated T cells irrespective of their antigenic specificity appears to hold promise in terms of its effectiveness and relative paucity of side effects. Both therapies were evaluated in the animal model and showed positive therapeutic effects. Future plans are to expand this approach to a larger number of patients.

2. Immune mechanisms in uveitis Uveitis is the most common intraocular inflammation. It results from disruption of the blood-ocular barrier and is characterized by leukocyte infiltration and protein leakage. Although the etiology and pathogenesis of uveitis are not well elucidated, there are several animal models for understanding disease pathogenesis and testing new therapies for ocular inflammatory diseases, such as interleukin (IL)-1 induced uveitis, experimental autoimmune uveitis (EAU), endotoxin induced uveitis (EIU), etc [2–6]. Among these models, IL-1 induced experimental uveitis is considered to be an animal model for acute anterior ocular inflammation. It is also induced in experimental animals by immunization with retinal antigens or their fragments, or by infusion of T cells specific to these antigens. Although no animal model fully reproduces the broad spectrum of human uveitic disease, the EAU model appears to share many essential features and mechanisms with human uveitis. Importantly, the EAU model has allowed the cellular mechanisms involved in the pathogenesis of autoimmune ocular disease to be studied at a level that would not be possible in human patients. This knowledge is critical for devising novel approaches to therapy, based on specifically targeting the processes and cells that participate in initiating and orchestrating the disease process. EIU is an acute anterior uveitis of short duration that is not autoimmune. EIU is induced in rats and mice by systemic injection of endotoxin from gram-negative bacteria. Although it is not clear whether there is an equivalent disease entity in humans, this model has been extremely useful in studying various aspects of ocular inflammation and in evaluating therapeutic interventions. Intraocular inflammatory disease, or uveitis, appears to be due in large part to noninfectious, cell-mediated mechanisms. The use of animal models has been very useful in better understanding mechanisms of ocular disease and bringing new therapeutic approaches to the clinic [7]. 3. Immunomodulation Based on an understanding of the critical checkpoints in the disease process, two major approaches to immunomodulation have emerged which seek to target the autopathogenic T cells that orchestrate the disease process: antigen specific, versus non-antigen specific. The antigen specific approach targets the disease related T cells based on their receptor for antigen (T cell receptor). For this approach to be successful, the antigen that drives the disease must be known. Antigens that may be involved in ocular autoimmune diseases in humans are increasingly being identified. The best known is retinal S-antigen, to which many uveitis patients exhibit lymphocyte activation responses. This antigen was shown to elicit typical uveitis in human leukocyte antigen transgenic mice, a “humanized” model for uveitis, supporting

4. Various therapeutic approaches for uveitis Immunomodulation therapy is increasingly being used in various ocular inflammatory diseases, to avoid the effects of long-term steroid and immunosuppressive therapy and to induce remission in chronic disease. This therapy seems to be effective specially in treating resistant disease with relative safety. New approaches to therapy of intraocular inflammatory diseases will emphasize less immunosuppressive drugs and modification of the immune system with anti-major histocompatibility complex II, anti-adhesion molecules, anti-cytokines monoclonal antibodies, therapy with cytokines antagonists, intravenous immunoglobulin therapy, tumor necrosis factor (TNF)-α therapy, antigen specific tolerating therapy and gene therapy. Advances in the understanding of pathogenesis and in diagnostic techniques in various ocular inflammatory entities, will serve as a driving force for these new methods of treatment. 5. Chemokines and their role in immunotherapy for intraocular inflammation Chemotactic cytokines are responsible for leukocyte migration and the immunopathogenesis of various inflammatory lesions. Together with other types of cytokines, chemokines play a major role in inducing/regulating inflammation and various immune responses. By targeting chemokines, immunotherapies could become another option for treating patients with uveitis. Indeed, a variety of chemokine based therapies have been tested for their possible application for various pathological diseases, including intraocular inflammation. An example of chemokine based therapy is anti-TNF-α therapy, a very successful treatment. Chemokine and cytokine based therapies, therefore, appear to be a promising choice for the treatment of intraocular inflammation. But, more effective and safer drugs need to be developed for improved future therapeutic use. Manipulations of cytokines expression by Th1 lymphocytes will be one of them [8,9]. Modulation of the cytokine network by either blocking cytokine activity or administration of regulatory Th2 cytokines has shown its efficacy in several experimental autoimmune diseases including uveitis. However, cytokines present pleiotropic activities and thus may exert different effects depending on the autoimmune diseases, making interventions on their production complex. Anti-cytokine therapy or a combination of anti-cytokine drugs, antibodies, and cytokine gene therapy to synergize the therapeutic effects of other treatments appear to be of interest. Improvements in drug delivery and in biotechnology will also allow us to elaborate new and safe immunomodulatory strategies.

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Anti-TNF-α antibodies have been used for treating inflammation in patients. Monoclonal antibodies against TNF-α are one of the candidates in giving us more opportunities to treat such patients more effectively. Its unique adverse side effects, though, should be carefully monitored. Data from animal experiments indicate that TNF-α is an important part of the proinflammatory processes in non-infectious uveitis. Neutralization of TNF-α with TNF receptor fusion proteins or monoclonal antibodies, therefore, represents a promising strategy for the treatment of uveitis. In addition, the currently available TNF-α inhibitors demonstrate a favorable profile of side effects compared to conventional immunosuppressive agents. Previous studies showed the immunological efficacy of TNF-α inhibitors in the treatment of posterior uveitis [9]. Anti-TNF-α treatment may be of value in the treatment of uveitis, and in patients with Behcet's disease, leading to suppression of ocular inflammation, vasculitis, and improvement of vision in the majority. Based on these results a controlled masked study is warranted. In animal experiments, TNF-α has been detected in an early phase of EIU in rats [10]. Increased levels of inflammatory cytokines such as TNF-α have been implicated in the pathogenesis of EAU in rats [11] and in mice [12]. This cytokine may induce the expression of chemokines, adhesion molecules, and other cytokines involved in the prolongation of inflammation. Inhibition of TNF-α activity results in suppression of Th1 effector mechanism, suppresses activation of infiltrating macrophages, and prevents tissue destruction in EAU [13,14]. Greiner et al. [15] showed that anti-TNF-α therapy modulates peripheral blood T cells in patients with posterior segment intraocular inflammation, which contributes to the recovery of visual function. In patients with uveitis including Behcet's disease, TNF-α levels are increased in serum and in aqueous humor [16,17]. Because of its pivotal role in inflammation, blockade of TNF-α activity may be effective in the treatment of uveitis. Anti-TNF-α therapies were originally established in rheumatoid arthritis [18]. Infliximab is a chimeric IgG monoclonal antibody directed against TNF-α. It binds with high affinity to the soluble and transmembrane forms of TNF-α [19]. Because of its impressive anti-inflammatory effects anti-TNF-α therapy has been successfully used in other inflammatory diseases like Crohn's disease [20], sarcoidosis [21], ankylosing spondylitis [22], and psoriatic arthritis [23,24]. In recent articles favorable results were reported in the treatment of patients with Behcet's disease [25] and also endogenous uveitis [26–28]. However, serious side effects have also been reported, including exacerbation of demyelinating disease [29], bilateral anterior optic neuropathy [30], tuberculosis [31], histoplasmosis [32], and even sudden death in patients with cardiac insufficiency. Anti-TNF-α therapy is promising in the treatment of severe sight threatening uveitis, both in patients with Behcet's disease and in idiopathic endogenous uveitis. Lindstedt EW et al. [33] reported their experience of infliximab treatment in a case series of 13 patients with sight threatening uveitis refractory to conventional immunosuppressive therapy. They included a series of patients in whom maximum visual recovery is limited as a result of irreversible ocular damage. Despite the poor recovery of visual acuity, that is, 7/13 patients showed improvement in vision, whereas 4/13 patients gained two or more lines, a good clinical response was observed regarding inflammation in the majority of these patients. Clinical trials are necessary to determine the optimal therapeutic strategy in patients with uveitis with regard to dosage and duration of treatment. They observed only minor side effects in two patients. In this small case series of refractory uveitis patients, they did not observe any signs or symptoms of lupus erythematosus. However, those patients were initially treated with other immunosuppressive drugs which may suppress autoantibody formation. The treatment of posterior uveitis is based on local or systemic immune suppression and the treatment of secondary macular edema.

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In most patients, treatment with steroids and cyclosporine reduces the inflammation sufficiently [1]. In a minority of patients other immunosuppressants, such as cytotoxic agents, are necessary as well. A novel approach is treatment with anti-TNF-α therapy. Behcet's disease is not a chronic, persistent inflammation, but rather a disorder characterized by recurrent attacks of acute inflammation. A rapid therapeutic effect is important in Behcet's disease in order to prevent ocular complications that result in irreversible visual impairment because of inflammation, macular edema, atrophy, or retinal detachment. Infliximab administration indeed results in a rapid decrease of the inflammation activity. Anti-TNF-α therapy may be added to the therapeutic regimen in the treatment of patients with sight threatening uveitis including uveitis in Behcet's disease. However, controlled masked studies are warranted to determine the optimal dosage and duration of the treatment. The effect of co-medication interaction must also be investigated in this new therapy for uveitis. In conclusion, antiTNF-α therapy is promising in the treatment of sight threatening uveitis. 6. Gene therapy and its role in uveitis The eye has unique advantages as a target organ for gene therapy of both inherited and acquired ocular disorders and offers a valuable model system for gene therapy. The eye is readily accessible to phenotypic examination and investigation of therapeutic effects in vivo by fundus imaging and electrophysiological techniques. Considerable progress has been made in the development of gene replacement therapies for retinal degenerations resulting from gene defects in photoreceptor cells and in retinal pigment epithelial cells using recombinant adeno-associated virus (AAV) and lentivirus-based vectors. Gene therapy also offers a potentially powerful approach to the treatment of complex acquired disorders such as those involving angiogenesis, inflammation and degeneration, by the targeted sustained intraocular delivery of therapeutic proteins. By directly introducing into ocular cell genes that encode proteins capable of down regulating the immune response, gene therapy has potential for both therapy and as a method for studying mechanisms of disease. While marked and rapid advances in the study of gene therapy have been realized, technical questions regarding the appropriate vector or the choice of efficacious immunomodulatory protein still remain. However, ocular gene therapy potentially offers advantages over recombinant cytokine or receptor fusion protein treatment as longterm expression of the therapeutic transgene negates the need for repeated systemic administration or potentially traumatic intraocular injections and administration of expensive recombinant cytokines. In addition, the ocular environment offers an excellent opportunity to study the potential of gene therapy strategies to modulate the immune response. Thus, gene therapy, either used alone or as an adjuvant, may provide an effective treatment for blinding ocular inflammatory disease. Gene therapy is a novel form of drug delivery that enlists the synthetic machinery of the patient's cells to produce a therapeutic agent. Genes may be delivered into cells in vitro or in vivo utilizing viral or non-viral vectors. Gene transfer into ocular tissues has been demonstrated with growing functional success and may develop into a new therapeutic tool for clinical ophthalmology in future. Gene therapy is a very attractive therapeutic option, as it carries the promise of more or less permanently curing a clinical condition. In the case of antigen specific therapy, arguably the most desirable approach, choice of antigen is a central issue, as in many cases the participating antigen is uncertain and multiple specificities may be involved. Not to be ignored also is the potential for eliciting unwanted immune responses by the introduction of an autoantigen into an already primed host. Paradigms targeting common lymphocyte activation functions have the potential of inhibiting desired immune responses as well, whereas strictly local therapies, while having the potential for the fewest side effects, leave the underlying autoimmune

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process unaffected. Gene therapy holds the promise of permanent cure through long-term expression of gene(s) designed to correct an underlying disease process. However, its success is highly dependent on the gene delivery system and in some cases on the ability to control gene expression [34]. Although gene therapy of retinal degeneration caused by single gene defects has been under investigation for some time, gene therapy applied to uveitis is still a new concept and, to date, only a few gene therapy studies in uveitis models have been attempted. However, the results have been promising. Fang IM et al. [35] conducted a study to investigate the efficiency of adenoviral-mediated transfer of the IL-10 gene for inhibition of experimental autoimmune anterior uveitis, a rat model of human acute anterior uveitis. Uveitis was induced in the Lewis rat by simultaneous intraperitoneal (i.p.) injection of melanin-associated antigen. The animals were treated by systemic administration of adenoviral construct expressing IL-10 (Ad-IL-10) or carrying no cytokine transgene. A significant reduction in ocular inflammation was observed for rats that received one or two divided administrations of Ad-IL-10, as assessed by reduced clinical scores and decreased leukocyte infiltration in the anterior chamber and confirmed by histological examinations, relative to control animals. Systemic Ad-IL10 treatment also revealed a higher serum level of IL-10 compared to the controls. Results of this study suggest that systemic adenovirusmediated IL-10 gene therapy has an anti-inflammatory effect on immune-mediated ocular inflammation. This approach may be promising for the treatment of acute anterior uveitis. Trittibach P et al. [36] investigated the effects of anterior chamber lentiviral vector delivery of genes encoding murine IL-1Ra (mIL-1Ra) and murine IL-10 (mIL-10) on the inflammatory response in a murine EIU model. Furthermore, the treated eyes showed less in vivo fluorescein leakage from inner retinal vessels compared with controls. The combination of both IL-1Ra and IL-10 had no additive effect. Thus, lentiviral gene delivery of IL-1Ra or IL-10 significantly reduces the severity of experimental uveitis, suggesting that lentiviral-mediated expression of immunomodulatory genes in the anterior chamber offers an opportunity to treat uveitis. Injection of lentiviral vectors into the anterior chamber of the eye, results in sustained expression of transgene in the anterior segment. Anterior chamber administration of a lentiviral vector may therefore enable sufficiently high level expression of immunomodulatory molecules at the site of inflammation to treat chronic anterior/intermediate uveitis without inducing systemic immunosuppression. Murine endotoxin induced uveitis is employed to assess the efficacy of immunotherapies as a surrogate for anterior and intermediate uveitis in man. They used this model to investigate the therapeutic effects of IL-1Ra and IL-10 using a lentiviral vector. They showed not only that anterior chamber lentiviral delivery of mIL-1Ra or mIL-10 significantly suppresses EIU but this inhibition of leukocyte infiltration into the posterior and anterior segments is also maintained in recurrent disease. Previous studies showed the ability of recombinant IL-10 to attenuate inflammatory conditions [35,37,38]. IL-10 has also been shown to downregulate ocular inflammation or to contribute to a higher threshold of resistance to uveitis using both recombinant protein [39] and gene transfer [35,40,41]. IL-1Ra has also demonstrated therapeutic efficacy in inflammatory conditions [42–44]. Although administration of recombinant cytokines can show therapeutic efficacy, many therapeutic regimens require daily administration due to the short half-life of the cytokine molecules and swift clearance from the circulation. Indeed, the serum half-life of recombinant IL-10 is between 2.3 and 3.7 h, and therefore the cytokine may be cleared before reaching the target organ [45]. Gene transfer techniques therefore offer several advantages over recombinant cytokine administration, including continuous production of therapeutic concentrations of cytokine and local expression of cytokine in target tissues. In vitro studies have shown that gene transfer of IL-1Ra is more effective than recombinant IL-1Ra at

inhibiting the biological actions of IL-1β in human synovial fibroblast cultures [46]. This study shows that significantly fewer infiltrating leukocytes were present in the Lenti IL-1Ra-treated eyes compared with controls. Several studies have previously shown the efficacy of IL-10 gene transfer using an AAV vector in another model of murine uveitis including the study using a tetracycline inducible system [40,47]. This study suggests that anterior chamber delivery of viral vectors expressing IL-1Ra and IL-10 can effectively treat murine experimental uveitis without resulting in systemic immune suppression, suggesting that intraocular delivery of immunomodulatory genes might offer an opportunity to develop more effective treatments for uveitis. Tsai ML et al. [48] conducted another study to evaluate the therapeutic potential of a recombinant adeno-associated virus vector encoding the IL-1 receptor antagonist gene (rAAV-IL-1Ra) in the treatment of experimental uveitis. To evaluate the therapeutic potential of rAAV-IL-1Ra, experimental uveitis was induced by intravitreal injection of IL-1α at 10 and 100 days after rAAV-IL-1Ra administration. Following intravitreal injection of rAAV-IL-1Ra, transgene expression was found in various cell types of the ocular tissues, such as ciliary epithelial cells, retinal ganglion cells, and retinal pigment epithelial cells. Long-term suppression of experimental uveitis could be achieved by gene therapy with rAAV-IL-1Ra. IL-1Ra is a member of the IL-1 family. Previous studies have reported that IL-1Ra improves many ocular inflammatory diseases, such as conjunctivitis and corneal graft rejection [49,50]. Experimental uveitis can also be suppressed by administration of IL-1Ra protein [51,52]. However, as the half-life of IL-1Ra protein is short (15–25 min), it is thus impractical for clinical uveitis therapy. Advances in molecular biology suggest that gene therapy with the IL-1Ra encoding gene has potential for the treatment of uveitis. If the IL-1Ra encoding gene can be delivered into the target cells and this results in the stable expression of that gene, such a strategy may overcome the problem of the short half-life of functional IL-1Ra protein. To date, naked DNA-, retroviral-, and adenovirus-based vectors have been studied as potential agents in ocular gene therapy [53,54]. However, these studies indicate that these vectors have some limitations in achieving ideal ocular gene therapy, including poor delivery efficiency, lack of sustained expression, and host immune reactions. The AAV is a promising gene delivery system because this vector might be used to deliver appropriate genes into diverse cell types in many tissues without causing significant host immune reactions. There are reports of recombinant AAV (rAAV) vectors that can deliver a transgene into the cells of various tissues, such as the central nervous system, muscle, lung, and gut, and produce long-term expression [55–58]. The rAAV vector encoding the human IL-1Ra encoding gene, which is driven by the human cytomegalovirus promoter, was introduced into the eyes of rabbits by intravitreal injection. The expression of the rAAV-mediated transgene in ocular tissue and the effects of rAAV-IL-1Ra on experimental uveitis were evaluated. Results suggest that experimental uveitis induced by IL-1 at 10 and 100 days after rAAV-IL-1Ra administration could be suppressed by a single intravitreal injection of rAAV-IL-1Ra. Previous studies have reported that IL-1Ra is a potential anti-inflammatory agent in the treatment of uveitis. IL-1Ra may suppress experimental uveitis by protecting the blood-ocular barrier from disruption by IL-1 stimulation. Although gene therapy with rAAV vectors is a promising approach in the treatment of experimental uveitis, there are still several improvements required to achieve practical management in the treatment of clinical uveitis in humans. Previous research has revealed that transgene expression decreases over time because most rAAVmediated transgenes are episomal, and only a few integrate randomly into the host genome [59,60]. Moreover, intravitreal administration of AAV vectors can elicit neutralizing antibodies against the vector capsid, thus decreasing the efficiency of therapeutic gene transfer and preventing effective vector readministration [61]. Thus, the stability of rAAV-mediated transgene expression remains a challenge.

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Furthermore, long-term constitutive transgene expression is not always desirable under certain conditions. In diseases such as intraocular inflammation, the level and timing of transgene expression must be regulated. Gene therapy with cell specific and inducible expression systems may offer an attractive strategy for obtaining therapeutic efficacy and avoiding toxicity. Additional modification of the AAV vector design might include a cell specific and inflammation inducible promoter system. In addition, various cell transduction and exogenous therapeutic gene products may compromise the functioning of the neuroretina. There is a theoretical risk associated with gene therapy with an AAV vector, although previous studies report that the rAAV vector is a safe gene delivery system [62]. Besides, clinical uveitis can be caused not only by IL-1 but also by other cytokines such as IL-6, IL-10, and tissue necrosis factor, etc [63–65]. 7. Gene therapy using immunoglobulins Immunoglobulins can serve as tolerogenic carriers for antigens, and B cells can function as tolerogenic antigen presenting cells. In 2000, Agarwal RK et al. [66] used this principle to design a strategy for gene therapy of EAU, a cell-mediated autoimmune disease model for human uveitis induced with the uveitogenic interphotoreceptor retinoid-binding protein (IRBP). In this study, they used a retroviral gene therapy strategy to prevent or reverse EAU, demonstrating that recipients of lipopolysaccharides (LPS) blasts transduced with a tolerogenic construct encoding the murine 161–180 peptide of IRBP in frame with murine IgG1 were highly protected from EAU induced with either the human or the murine uveitogenic peptide homologues and were significantly protected from challenge with the whole, multiepitope, IRBP molecule. They suggest that this form of gene therapy can induce epitope specific protection not only in naive, but also in already primed recipients, thus providing a protocol for treatment of established autoimmunity. This study is therefore an important advance that translates findings obtained in a model antigen system to a therapeutic setting. Previous studies have used intravenous infusions of antigens chemically coupled to autologous immunoglobulins or cells to induce tolerance [67–69]. The advantage of the present approach is that it introduces a self renewing source of tolerogen that establishes residence in the body and essentially becomes part of “self,” as opposed to remaining an exogenous treatment whose effects may be transient. Moreover, in already immune individuals the present approach has an additional advantage over an intravenous bolus of an antigen-Ig conjugate in that it avoids introducing a large amount of antigen into the bloodstream, which might trigger an anaphylactic reaction. There is also a clear advantage to using ex vivo transduced autologous cells over direct administration of the chimeric retrovirus, because it largely circumvents the problems inherent in administering immunogenic viral vectors in vivo. In summary, they demonstrated that gene therapy with autologous cells transduced with a retroviral construct composed of an uveitogenic epitope fused with an isologous IgG molecule can prevent as well as reverse EAU. The effectiveness of this therapy in preimmune as well as in naive recipients opens the possibility of using this approach in a clinical situation when the patient has a pre-existing repertoire of lymphocytes primed to an autologous antigen [34]. 8. RNA interference (RNAi) as therapeutic approach in uveitis It is of interest to mention RNA interference (RNAi), a relatively new technology for gene silencing that has been gaining momentum and is rapidly replacing the antisense DNA approach. Significant progress has been made in advancing RNAi therapeutics in a remarkably short period of time. Therapeutics based on RNAi offer a powerful method for rapidly identifying specific and potent inhibitors of disease targets from all molecular classes. Numerous proof-of-

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concept studies in animal models of human disease demonstrate the broad potential application of RNAi therapeutics. The major challenge for successful drug development is identifying delivery strategies that can be translated to the clinic. With advances in this area and the commencement of multiple clinical trials with RNAi therapeutic candidates, a transformation in modern medicine may soon be realized. In RNAi, the target mRNA is enzymatically cleaved, leading to decreased abundance of the corresponding protein, and specificity is a key feature of the mechanism. Synthetic small interfering RNAs (siRNAs) leverage the naturally occurring RNAi process in a manner that is consistent and predictable with regard to extent and duration of action. In addition, viral delivery of short hairpin RNAs (shRNAs) represents an alternative strategy for harnessing RNAi. Both non-viral delivery of siRNAs and viral delivery of shRNAs are being advanced as potential RNAi-based therapeutic approaches. Recent findings have highlighted the effectiveness of RNAi in therapeutically relevant settings, the results of which have spurred cautious optimism about the promise of RNAi-based therapies. The first clinical applications of RNAi have been directed at the treatment of wet age related macular degeneration and respiratory syncytial virus infection. Therapies based on RNAi are also in preclinical development for other viral diseases, neurodegenerative disorders and cancers, although a number of challenges need to be addressed and improvements made for RNAi-based therapies to realize their full potential. RNAi may find application in ocular therapeutics to inhibit expression of transcription factors or of proinflammatory cytokines instead of the currently used toxic pharmacological agents. Due to its complementary nature, it is highly specific to its target sequence, although as therapy one must take into account the possibility of crossreactive, off-target and nonspecific effects. However, the method of introducing the siRNA-generating DNA fragments into cells relies on the same viral and non-viral methods as “traditional” gene therapy, so in that regard similar caveats will apply. There are few studies, in which siRNAs has been used to explore its therapeutic potential in uveitis. In 2008, to inhibit the expression of TNF-α, Choi B et al. [70] used siRNAs to reduce overexpression of TNF-α in vitro in cell cultures and in an in vivo Behcet's disease-like mouse model for amelioration of chronic inflammation. TNF-α siRNA was injected i.p. twice with a one week interval. To compare the efficacy of TNF-α siRNA versus an anti-TNF-α antibody, infliximab and etanercept were administered to symptomatic mice with inflamed tissue. Intraperitoneal delivery of TNF-α siRNA effectively decreased Behcet's disease-like symptoms in 18 of 32 cases (56.3%). Scrambled siRNA treatment decreased Behcet's disease-like symptoms in 2 of 19 cases (10.5%). Infliximab was effective in 11 of 27 cases (40.7%) and etanercept was also effective in 9 of 25 cases (36%) at the end of the second week after treatment. TNF-α siRNA reduced serum levels of TNFα compared to levels in mice not injected or scramble injected. These results suggested that siRNAs can be employed to inhibit cytokine gene expression in an in vivo disease mouse model. This inhibition may, therefore, be attributed to the improvement of inflammatory symptoms. Recently, it has been seen that inducible co-stimulator (ICOS) was up-regulated in experimental autoimmune uveoretinitis. Another study has been conducted to investigate whether intravitreal injection siRNA plasmid, targeting ICOS, suppresses the ongoing experimental autoimmune uveoretinitis in rats [71]. The recombinant plasmid (pRNAT-U6.1/ Neo-ICOS) for the ICOS siRNA was successfully constructed. In vitro studies using the recombinant plasmid has showed the down regulation of ICOS gene expression both at the mRNA and protein levels. Clinical and pathological scores showed that ocular inflammation of pRNATU6.1/Neo-ICOS-treated eyes was markedly less than that of vehicletreated eyes. The expression of ICOS protein and the amount of CD4(+) ICOS(+) T cells in retina significantly decreased by intravitreal injection of the recombinant plasmid, whereas delayed type hypersensitivity response and lymphocyte proliferation were not impaired in rats treated with the recombinant plasmid. Intravitreal injection of siRNA plasmid targeting ICOS effectively down regulated the expression of

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ICOS, and was highly effective in suppressing the ongoing process of autoimmune uveoretinitis without any side effects on systemic cellular immunity. Although it is quite evident that in the management of intraocular inflammation immunosuppression by steroids and cytotoxic agents may be supplanted by a new range of immunomodulators, these new drugs need to be tested in human uveitic conditions. The results from the animal model cannot be applied directly to human diseases. Side effects of these new molecules need to be weighed against the standard therapy. Last, but not least, the cost of such therapy needs to be evaluated. Gene therapy is a very attractive therapeutic option, as it carries the promise of more or less permanently curing a clinical condition. In conclusion, as our understanding of the critical checkpoints in the pathogenesis of autoimmune ocular disease improves, more and more potential intervention points and candidate therapeutic targets are identified. New technologies emerge, promising more specific and more easily applied therapies. Nevertheless, serious concerns about vector development and delivery methods remain to be addressed, including such issues as vector immunogenicity, method of administration, efficiency of transduction, duration of gene expression as well as the ability to turn expression off when deemed necessary. None of the therapy paradigms offers a perfect solution. The initial step is therefore to understand the immunobiology of intraocular inflammation which will provide insight into the current advancements in therapeutic research in uveitis and will prepare us for newer therapies in uveitis.

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