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Antisense approaches to immune modulation for transplant and autoimmune diseases
Antisense approaches to immune modulation for transplant and autoimmune diseases Dan V Mourich and Nikki B Marshall Antisense oligomers have been shown to be effective tools for inhibiting gene expression in a highly specific manner. This technology has proven to be invaluable for determining gene function in conventional molecular and cellular studies. However, the promise of an antisense-based drug technology, suggested by antiviral efficacy shown nearly 25 years ago, is just now coming of age. Since then, numerous antisense approaches have been shown to be effective in animal models against numerous viruses and some tumors. Not surprisingly, antisense agents targeting these diseases are taking the lead in human clinical trials and FDA approval. Although comparatively smaller in scope, approaches for modulating immune responses to treat Crohn’s disease, diabetes, multiple sclerosis and transplant rejection appear to be the next burgeoning phase of development in antisense therapy. Addresses Oregon State University Department of Microbiology, Nash Hall 220, Corvallis, OR 97333, USA Corresponding author: Mourich, Dan V ([email protected])
Current Opinion in Pharmacology 2005, 5:508–512 This review comes from a themed issue on New technologies Edited by Patrick Iversen Available online 8th August 2005 1471-4892/$ – see front matter # 2005 Elsevier Ltd. All rights reserved.
as result of autoimmunity comprise the same elements. First, leukocytes (white cells of the innate and adaptive immune systems) must gain access to the tissue in question. Once established in the periphery, inflammatory processes ensue and autoantigens (self) or alloantigens (non-self) are revealed to the adaptive immune system. Infiltrating leukocytes then produce cytokines and chemokines, facilitating the trafficking of activated antigenspecific effector T lymphocytes to the site. These cells in turn secrete or express on their surface effector proteins that either cause direct cellular destruction or act to amplify the inflammatory process further. Oversimplified as the above description of the immune response might be, it is plausible that targeted inhibition of proteins essential to any part of these processes could serve as an effective immunosuppressive therapy. Generally speaking, any therapeutic approach designed to subdue the immune response, including antisense drugs, can be divided into three crucial points of intervention: reduction or inhibition of the inflammatory process; inhibition of the activation or function of antigen-specific lymphocytes; or promotion of active tolerance to tissuespecific antigens (Figure 1). Drugs which successfully suppress these responses, although not without numerous side effects, have made possible the transplantation of tissues and solid organs and the alleviation of painfully destructive inflammation. Although at widely ranging stages of development, each of these different approaches is being pursued with antisense agents [3,4].
DOI 10.1016/j.coph.2005.04.018
Antisense targets for autoimmunity and transplant survival Introduction
Adhesion molecules
Antisense molecules bind to DNA or RNA through classic Watson and Crick base paring and can be used to alter protein function or inhibit expression. Several mechanisms of action have been demonstrated, including steric blockade of translation initiation and progress, altered pre-mRNA splicing or RNase-mediated degradation of message. The mode of action is, in part, determined by the chemical composition of the oligomer backbone and linkages [1]. Many different chemical modifications have been developed to improve cellular delivery, stability and efficacy without compromising specificity [2]. It is the latter characteristic that makes antisense therapy an attractive platform for developing agents to modulate the immune system.
The general movement of leukocytes is accomplished by the force of blood flowing through the vasculature and is controlled by a process of rolling adhesion along the vascular endothelium. Directed trafficking is accomplished through progressively tighter interactions between selectins and integrins expressed on leukocytes and corresponding adhesion ligands expressed by endothelial cells. Together, these create a ‘tether’ capable of holding the cell against the shear forces produced in the blood vessel. Various proinflammatory signals can increase the expression of these molecules and thus bring about strong adhesion at or near the site of inflammation. When rolling is arrested, owing to sufficient adhesion, leukocytes enter the surrounding tissue by squeezing between the endothelial junctions in the vessel.
The rudimentary immune responses involved during the rejection of grafted tissue or destruction of healthy tissue
The migration and homing of leukocytes to sites of inflammation is absolutely crucial for any subsequent
Current Opinion in Pharmacology 2005, 5:508–512
www.sciencedirect.com
Antisense approaches to immune modulation for transplant and autoimmune diseases Mourich and Marshall
509
Figure 1
Blood flow
X X X X
Mθ
(a)
APC
T cell
T cell
X
X X
Treg
X (b)
(c) Current Opinion in Pharmacology
Points of intervention for immunosuppressive therapeutics. (a) Inflammatory processes can be reduced by inhibiting expression of leukocyte adhesion molecules (blue diamonds) or the production of proinflammatory cytokines and chemokines (blue circles) or their cognate receptors (black bars). Decreased adhesion or response to chemotactic signals does not allow inflammatory cells to overcome the shear forces of blood flow in the vessel wall (blue horizontal blocks). (b) T cell activation or effector function can be altered by inhibiting expression of co-stimulatory molecules (black/white blocks on cell surfaces), cytokines produced by antigen-presenting cells (APCs; blue circles) or T cell cytokine receptors (grey block), or effector molecules or receptors produced by T cells that cause direct tissue destruction (white circles and black diamonds, respectively). (c) T regulatory (Treg) cells can protect tissues from auto-aggressive T cell destruction by producing immunosuppressive cytokines (black circles). The red crosses indicate the specific molecules to be targeted for inhibited expression.
immune responses to take place in peripheral tissue. Therefore, it is not surprising that several therapeutic approaches have been, and are being, developed to target adhesion molecules expressed on leukocytes or endothelium for treatment of inflammatory diseases and transplant survival. Moreover, this strategy is currently leading the field of antisense drug development for the treatment of such autoimmune diseases as Crohn’s disease (CD), ulcerative colitis and psoriasis [2,4]. Additionally, recent studies show promising results for this approach in an animal model for multiple sclerosis (MS) [5]. ICAM-1
Intercellular adhesion molecule 1 (ICAM-1), a member of the immunoglobulin superfamily, is constitutively expressed at low levels on vascular and colonic endothelium. Surface expression is increased in response to various inflammatory stimuli, increasing the adhesion of monocytes, macrophages, neutrophils, T lymphocytes and dendritic cells (DCs) expressing the integrin molecules lymphocyte function-associated antigen 1 and/or Mac1. Theoretically, inhibiting the expression or function of ICAM-1 would hinder migration of all of these cell types and thus influence both general inflammatory and www.sciencedirect.com
antigen-specific T cell responses, resulting in a broad and potent immunosuppressive effect. To date, clinical trials examining the therapeutic potential of an anti-ICAM agent have been solely conducted using antisense oligonucleotides. Phase II and III clinical trials for the treatment of steroid-dependent and steroidrefractory Crohn’s disease have been carried out using alicaforsen (ISIS Pharmaceuticals), a 20-base oligomer complementary to the human ICAM-1 mRNA, utilizing the phosphorothioate backbone. Although no therapeutic benefit was demonstrated against steroid-refractory CD, the results in patients under steroid-dependent treatment were evident. It should be noted that success was marginal because the primary endpoint of the study, steroidfree remission, was not achieved but the disease activity index did improve for those patients receiving combination therapy [6]. In a trial examining escalating doses of enema-administered alicaforsen in patients with mild to moderate ulcerative colitis, a dose-dependent benefit was seen for acute and long-term disease activity [7]. Taken together, these studies suggest some benefit of antiICAM therapy for inflammatory bowel diseases; however, it is likely that modifications to the current antisense chemistry, routes of administration and dose regimen will Current Opinion in Pharmacology 2005, 5:508–512
510 New technologies
need to be considered before this approach can become a stand-alone therapy. Patients receiving a kidney transplant are often prone to developing renal ischemia-reperfusion (I-R) injury, initiated by localized renal damage due to reperfusion. The subsequent inflammatory response and leukocyte infiltration can result in extensive tissue damage and, ultimately, acute renal failure. As yet, there is no effective treatment to prevent possible renal damage after ischemia or to control subsequent damage caused by inflammation. The mechanism behind I-R is not fully understood, yet several studies have demonstrated that the ensuing renal damage is mediated in large part by the adhesion of neutrophils and lymphocytes to the vascular endothelium of the graft through ICAM-1 interactions. Two recent studies, using a syngenic renal transplant animal model, demonstrated that antisense targeting of ICAM-1 expression is helpful in preventing I-R injury [8,9]. Both studies employed liposome-complexed phosphorothioate oligomers targeting the 30 end of the ICAM-1 mRNA. One study paired the oligomer with FTY 720 (2-amino-2-[2-(4-octylphenyl) ethyl] propane1,3-diol hydrochloride), an analog of sphingosine which affects the leukocyte response to chemokines, whereas the other examined the influence of the oligomer alone [8]. In both cases, antisense oligonucleotides to ICAM-1 demonstrated a reduction in leukocyte infiltration, as seen by histological examination, and improved renal function compared with animals receiving no treatment or a control oligonucleotide. Furthermore, a mild additive effect was seen when the antisense oligonucleotide was paired with FTY 720 [9]. Although I-R can be induced independently of antigen-specific responses, in the clinic allogenic transplants are likely to be the rule, rather than the exception. For anti-ICAM therapy to be useful, it would need to be effective under these conditions. Application to an allograft was recently shown to work in a Lewis donor into a renal damageresistant AugustXCopenhagen Irish recipient rat model using a methoxyethyl-modified phosphorothioate to ICAM-1 [10]. Both intravenous and oral delivery of the antisense oligonucleotide did extend kidney graft survival, indicating that anti-ICAM-1 could be effective in the clinic. VLA-4
VLA-4 integrin is a heterodimeric molecule, consisting of an a4 and a b1 chain, expressed on most leukocytes and is involved in leukocyte trafficking into a variety of organs. Monoclonal antibodies specific for the a4 chain have proven to be effective in suppressing autoimmune disease in several animal models, and very early results from clinical trials for the treatment of CD and MS held great hope for a successful therapeutic agent. Antisense oligonucleotides to the a4 chain administered as a prophylactic Current Opinion in Pharmacology 2005, 5:508–512
or therapeutic treatment also work well in mouse experimental autoimmune encephalomyelitis (EAE) experiments [5].
Caveats to targeting adhesion molecules Leukocyte adhesion deficiency caused by specific defects in lymphocyte function-associated antigen 1 and Mac1 in humans or knockout mice results in recurrent bacterial infections and impaired wound healing. Even though indications of such complications have not emerged in any ICAM-1 trials, significant side effects have been reported in trials targeting VLA-4 with antibody. The US FDA had granted accelerated approval of the anti-VLA-4 (natalizumab) in November 2004 after reviewing a single year’s worth of data from two MS Phase III studies. Additionally, initial results in CD looked equally promising. However, after only a few months, the manufacturers of natalizumab voluntarily suspended the drug on 28 February 2005, owing to adverse effects that occurred in two patients treated with natalizumb along with interferon b1a, with two confirmed deaths [11]. These adverse events, including the deaths, were found to be due to progressive multifocal leukoencephalopahty (PML). In both cases, indications of a JC virus infection, a causative agent of PML, were observed. It is plausible that reduced immune surveillance brought on by the therapy led to the reactivation of latent virus. In light of these recent events, further development of antisense antiadhesion therapy will have to proceed with caution or be reevaluated.
Soluble mediators of inflammation and receptors Cytokines and chemokines can facilitate the homing and activation of lymphocytes and have a major role in inflammatory disease and graft rejection. Antisense studies targeting expression of these molecules or their cognate receptors have been performed in several animal models. In one study, antisense oligonucleotides to tumor necrosis factor were found to be effective in ameliorating acute and chronic inflammatory bowel disease in two different mouse models [12]. Atopic dermatitis, a human inflammatory disease of the skin, is, in part, caused by the overexpression of interleukin 10 (IL-10) by monocytes and macrophages in the dermis. Anti-IL-10 oligomers delivered by iontophoresis did cause a decrease in IL10 in skin lesions of mice, and a concomitant therapeutic benefit to established dermatitis [13]. Chemokines are the principal factors for directing T cells to allografted tissue. Several different chemokines can bind to the CXCR3 receptor expressed on memory and activated T cells to promote the rejection of skin allografts. Peptide nucleic acid antisense oligomers targeting CXCR3 expression were found to be effective in prolonging the grafts in mice by inhibiting the migration of T cells to the graft [14]. Moreover, this did not interfere with other T cell functions. www.sciencedirect.com
Antisense approaches to immune modulation for transplant and autoimmune diseases Mourich and Marshall
Co-stimulatory molecules and T regulatory cells The goal of an ‘ultimate’ immunosuppressive therapy would be to produce a state of tolerance to transplant tissue antigens or autoantigens. Controlling inflammation might, at best, have only a transient effect and require constant application of the antisense agent. DCs have a key role in initiating some mechanisms of antigen-specific tolerance. Manipulation of DC function (‘engineered DCs’) has been shown to be an effective method for inducing tolerance in autoimmune and transplant models [15]. Phenotypically ‘immature’ DCs express low levels of major histocompatibility complex molecules and costimulatory ligands (CD80, CD86 and CD40). DC maturation is induced by inflammatory signals, resulting in increased expression of these molecules, which are crucial for T cell activation [16,17]. Therapeutic administration of immature DCs can result in deletion of responding T cells and therefore tolerance through nonresponsiveness. A form of active tolerance can also be induced by immature DCs, whereby responding T cells take on a suppressive phenotype. These naturally occurring cells are referred to as regulatory T cells and possess very potent mechanisms for suppressing autoaggressive immune responses [18]. Antisense targeting of co-stimulatory molecules has been applied to both type 1 diabetes mellitus and allograft acceptance. Using a heterotopic heart transplant model in mice and DCs treated with antisense oligonucleotide to CD80 and CD86, it was shown that graft survival could be prolonged through depletion of allospecific T cells [19]. In the NOD mouse model, DCs treated with antisense oligonucleotides to CD40, CD80 and CD86 promoted the expansion of a regulatory T cell population and significantly delayed the onset of diabetes [20]. Co-stimulatory molecule targeting has shown some success when antisense oligonucleotides are administered to the whole animal [21] but only a single injection of antisense-modified DCs was necessary to produce tolerance in the two studies above [19,20], confirming the remarkable utility of ex vivo antisense therapy.
Conclusions It is clear that the development of antisense-based therapeutics is expanding into nearly all areas of human disease. The immune system offers a vast frontier of possibilities for targeted manipulation either to suppress unwanted responses or to augment those that could be beneficial. The inherent specificity of mRNA sequences offers researchers, through antisense, an unambiguous platform for drug design. Molecules with unique roles in specific immune-related diseases are continuously being defined [22,23,24]. As much needed improvements are made to the delivery of antisense oligonucleotides into immune cells, the development of numerous therapeutic agents will certainly follow. www.sciencedirect.com
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Acknowledgements Special thanks to Marta Johnson for her help in gathering literature which has been crucial to the writing of this manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Crooke ST: Progress in antisense technology. Annu Rev Med 2004, 55:61-95.
2.
Kurreck J: Antisense technologies. Improvement through novel chemical modifications. Eur J Biochem 2003, 270:1628-1644.
3.
Praditpornsilpa K, Avihingsanon Y: New concepts in organ transplantation. Transplant Proc 2004, 36:1228-1231.
4.
Van Assche G, Rutgeerts P: Physiological basis for novel drug therapies used to treat the inflammatory bowel diseases. I. Immunology and therapeutic potential of antiadhesion molecule therapy in inflammatory bowel disease. Am J Physiol Gastrointest Liver Physiol 2005, 288:G169-G174.
5.
Myers KJ, Witchell DR, Graham MJ, Koo S, Butler M, Condon TP: Antisense oligonucleotide blockade of alpha 4 integrin prevents and reverses clinical symptoms in murine experimental autoimmune encephalomyelitis. J Neuroimmunol 2005, 160:12-24. This study provided the first demonstration of the use of antisense oligonucleotide as a therapeutic treatment after onset of disease in this murine model for MS. 6.
Yacyshyn BR, Chey WY, Goff J, Salzberg B, Baerg R, Buchman AL, Tami J, Yu R, Gibiansky E, Shanahan WR: Double blind, placebo controlled trial of the remission inducing and steroid sparing properties of an ICAM-1 antisense oligodeoxynucleotide, alicaforsen (ISIS 2302), in active steroid dependent Crohn’s disease. Gut 2002, 51:30-36.
7.
van Deventer SJ, Tami JA, Wedel MK: A randomised, controlled, double blind, escalating dose study of alicaforsen enema in active ulcerative colitis. Gut 2004, 53:1646-1651.
8.
Kiew LV, Munavvar AS, Law CH, Azizan AN, Nazarina AR, Sidik K, Johns EJ: Effect of antisense oligodeoxynucleotides for ICAM-1 on renal ischaemia-reperfusion injury in the anaesthetised rat. J Physiol 2004, 557:981-989.
9.
Ortiz AM, Troncoso P, Kahan BD: Prevention of renal ischemic reperfusion injury using FTY 720 and ICAM-1 antisense oligonucleotides. Transplant Proc 2003, 35:1571-1574.
10. Chen W, Langer RM, Janczewska S, Furian L, Geary R, Qu X, Wang M, Verani R, Condon T, Stecker K et al.: Methoxyethylmodified intercellular adhesion molecule-1 antisense phosphorothiateoligonucleotides inhibit allograft rejection, ischemic-reperfusion injury, and cyclosporine-induced nephrotoxicity. Transplantation 2005, 79:401-408. This study provided the first demonstration that anti-adhesion therapy could prolong allograft survival using antisense oligonucleotides. 11. Sheridan C: Third Tysabri adverse case hits drug class. Nat Rev Drug Discov 2005, 4:357-358. This provides a detailed account of the circumstances leading up to the withdrawal of anti-VLA-4 antibody therapy. 12. Myers KJ, Murthy S, Flanigan A, Witchell DR, Butler M, Murray S, Siwkowski A, Goodfellow D, Madsen K, Baker B: Antisense oligonucleotide blockade of tumor necrosis factor-alpha in two murine models of colitis. J Pharmacol Exp Ther 2003, 304:411-424. 13. Sakamoto T, Miyazaki E, Aramaki Y, Arima H, Takahashi M, Kato Y, Koga M, Tsuchiya S: Improvement of dermatitis by iontophoretically delivered antisense oligonucleotides for interleukin-10 in NC/Nga mice. Gene Ther 2004, 11:317-324. 14. Jiankuo M, Xingbing W, Baojun H, Xiongwin W, Zhuoya L, Ping X, Yong X, Anting L, Chunsong H, Feili G, Jinquan T: Peptide nucleic Current Opinion in Pharmacology 2005, 5:508–512
512 New technologies
acid antisense prolongs skin allograft survival by means of blockade of CXCR3 expression directing T cells into graft. J Immunol 2003, 170:1556-1565. 15. Steinman RM, Hawiger D, Nussenzweig MC: Tolerogenic dendritic cells. Annu Rev Immunol 2003, 21:685-711. 16. Quezada SA, Jarvinen LZ, Lind EF, Noelle RJ: CD40/CD154 interactions at the interface of tolerance and immunity. Annu Rev Immunol 2004, 22:307-328. 17. Carreno BM, Collins M: The B7 family of ligands and its receptors: new pathways for costimulation and inhibition of immune responses. Annu Rev Immunol 2002, 20:29-53. 18. von Boehmer H: Mechanisms of suppression by suppressor T cells. Nat Immunol 2005, 6:338-344. This is a concise and comprehensive review of regulatory T cell mechanisms and molecules involved in their derivation and immune suppressive activity. 19. Liang X, Lu L, Chen Z, Vickers T, Zhang H, Fung JJ, Qian S: Administration of dendritic cells transduced with antisense oligodeoxyribonucleotides targeting CD80 or CD86 prolongs allograft survival. Transplantation 2003, 76:721-729. 20. Machen J, Harnaha J, Lakomy R, Styche A, Trucco M, Giannoukakis N: Antisense oligonucleotides down-regulating costimulation confer diabetes-preventive properties to
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nonobese diabetic mouse dendritic cells. J Immunol 2004, 173:4331-4341. This was both the first demonstration of an ex vivo antisense treatment to inhibit autoimmune disease, and the first to demonstrate that any antisense treatment could be used to elicit a T regulatory cell response. 21. Gao D, Wagner AH, Fankhaenel S, Stojanovic T, Schweyer S, Panzner S, Hecker M: CD40 antisense oligonucleotide inhibition of trinitrobenzene sulphonic acid induced rat colitis. Gut 2005, 54:70-77. 22. Lovett-Racke AE, Rocchini AE, Choy J, Northrop SC, Hussain RZ, Ratts RB, Sikder D, Racke MK: Silencing T-bet defines a critical role in the differentiation of autoreactive T lymphocytes. Immunity 2004, 21:719-731. This study demonstrated the first antisense targeting of a phenotypic differentiation factor in T cells. 23. Mayne M, Moffatt T, Kong H, McLaren PJ, Fowke KR, Becker KG, Namaka M, Schenck A, Bardoni B, Bernstein CN, Melanson M: CYFIP2 is highly abundant in CD4+ cells from multiple sclerosis patients and is involved in T cell adhesion. Eur J Immunol 2004, 34:1217-1227. This study demonstrated a potentially novel target for treating MS. 24. Ansari MA, Dhar M, Muthukrishnan V, Morton TL, Bakht N, Jacobson JD: Administration of antisense oligonucleotides to Galpha(Q/11) reduces the severity of murine lupus. Biochimie 2003, 85:627-632.
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Current Opinion in Pharmacology 2005, 5:508–512 This review comes from a themed issue on New technologies Edited by Patrick Iversen Available online 8th August 2005 1471-4892/$ – see front matter # 2005 Elsevier Ltd. All rights reserved.
as result of autoimmunity comprise the same elements. First, leukocytes (white cells of the innate and adaptive immune systems) must gain access to the tissue in question. Once established in the periphery, inflammatory processes ensue and autoantigens (self) or alloantigens (non-self) are revealed to the adaptive immune system. Infiltrating leukocytes then produce cytokines and chemokines, facilitating the trafficking of activated antigenspecific effector T lymphocytes to the site. These cells in turn secrete or express on their surface effector proteins that either cause direct cellular destruction or act to amplify the inflammatory process further. Oversimplified as the above description of the immune response might be, it is plausible that targeted inhibition of proteins essential to any part of these processes could serve as an effective immunosuppressive therapy. Generally speaking, any therapeutic approach designed to subdue the immune response, including antisense drugs, can be divided into three crucial points of intervention: reduction or inhibition of the inflammatory process; inhibition of the activation or function of antigen-specific lymphocytes; or promotion of active tolerance to tissuespecific antigens (Figure 1). Drugs which successfully suppress these responses, although not without numerous side effects, have made possible the transplantation of tissues and solid organs and the alleviation of painfully destructive inflammation. Although at widely ranging stages of development, each of these different approaches is being pursued with antisense agents [3,4].
DOI 10.1016/j.coph.2005.04.018
Antisense targets for autoimmunity and transplant survival Introduction
Adhesion molecules
Antisense molecules bind to DNA or RNA through classic Watson and Crick base paring and can be used to alter protein function or inhibit expression. Several mechanisms of action have been demonstrated, including steric blockade of translation initiation and progress, altered pre-mRNA splicing or RNase-mediated degradation of message. The mode of action is, in part, determined by the chemical composition of the oligomer backbone and linkages [1]. Many different chemical modifications have been developed to improve cellular delivery, stability and efficacy without compromising specificity [2]. It is the latter characteristic that makes antisense therapy an attractive platform for developing agents to modulate the immune system.
The general movement of leukocytes is accomplished by the force of blood flowing through the vasculature and is controlled by a process of rolling adhesion along the vascular endothelium. Directed trafficking is accomplished through progressively tighter interactions between selectins and integrins expressed on leukocytes and corresponding adhesion ligands expressed by endothelial cells. Together, these create a ‘tether’ capable of holding the cell against the shear forces produced in the blood vessel. Various proinflammatory signals can increase the expression of these molecules and thus bring about strong adhesion at or near the site of inflammation. When rolling is arrested, owing to sufficient adhesion, leukocytes enter the surrounding tissue by squeezing between the endothelial junctions in the vessel.
The rudimentary immune responses involved during the rejection of grafted tissue or destruction of healthy tissue
The migration and homing of leukocytes to sites of inflammation is absolutely crucial for any subsequent
Current Opinion in Pharmacology 2005, 5:508–512
www.sciencedirect.com
Antisense approaches to immune modulation for transplant and autoimmune diseases Mourich and Marshall
509
Figure 1
Blood flow
X X X X
Mθ
(a)
APC
T cell
T cell
X
X X
Treg
X (b)
(c) Current Opinion in Pharmacology
Points of intervention for immunosuppressive therapeutics. (a) Inflammatory processes can be reduced by inhibiting expression of leukocyte adhesion molecules (blue diamonds) or the production of proinflammatory cytokines and chemokines (blue circles) or their cognate receptors (black bars). Decreased adhesion or response to chemotactic signals does not allow inflammatory cells to overcome the shear forces of blood flow in the vessel wall (blue horizontal blocks). (b) T cell activation or effector function can be altered by inhibiting expression of co-stimulatory molecules (black/white blocks on cell surfaces), cytokines produced by antigen-presenting cells (APCs; blue circles) or T cell cytokine receptors (grey block), or effector molecules or receptors produced by T cells that cause direct tissue destruction (white circles and black diamonds, respectively). (c) T regulatory (Treg) cells can protect tissues from auto-aggressive T cell destruction by producing immunosuppressive cytokines (black circles). The red crosses indicate the specific molecules to be targeted for inhibited expression.
immune responses to take place in peripheral tissue. Therefore, it is not surprising that several therapeutic approaches have been, and are being, developed to target adhesion molecules expressed on leukocytes or endothelium for treatment of inflammatory diseases and transplant survival. Moreover, this strategy is currently leading the field of antisense drug development for the treatment of such autoimmune diseases as Crohn’s disease (CD), ulcerative colitis and psoriasis [2,4]. Additionally, recent studies show promising results for this approach in an animal model for multiple sclerosis (MS) [5]. ICAM-1
Intercellular adhesion molecule 1 (ICAM-1), a member of the immunoglobulin superfamily, is constitutively expressed at low levels on vascular and colonic endothelium. Surface expression is increased in response to various inflammatory stimuli, increasing the adhesion of monocytes, macrophages, neutrophils, T lymphocytes and dendritic cells (DCs) expressing the integrin molecules lymphocyte function-associated antigen 1 and/or Mac1. Theoretically, inhibiting the expression or function of ICAM-1 would hinder migration of all of these cell types and thus influence both general inflammatory and www.sciencedirect.com
antigen-specific T cell responses, resulting in a broad and potent immunosuppressive effect. To date, clinical trials examining the therapeutic potential of an anti-ICAM agent have been solely conducted using antisense oligonucleotides. Phase II and III clinical trials for the treatment of steroid-dependent and steroidrefractory Crohn’s disease have been carried out using alicaforsen (ISIS Pharmaceuticals), a 20-base oligomer complementary to the human ICAM-1 mRNA, utilizing the phosphorothioate backbone. Although no therapeutic benefit was demonstrated against steroid-refractory CD, the results in patients under steroid-dependent treatment were evident. It should be noted that success was marginal because the primary endpoint of the study, steroidfree remission, was not achieved but the disease activity index did improve for those patients receiving combination therapy [6]. In a trial examining escalating doses of enema-administered alicaforsen in patients with mild to moderate ulcerative colitis, a dose-dependent benefit was seen for acute and long-term disease activity [7]. Taken together, these studies suggest some benefit of antiICAM therapy for inflammatory bowel diseases; however, it is likely that modifications to the current antisense chemistry, routes of administration and dose regimen will Current Opinion in Pharmacology 2005, 5:508–512
510 New technologies
need to be considered before this approach can become a stand-alone therapy. Patients receiving a kidney transplant are often prone to developing renal ischemia-reperfusion (I-R) injury, initiated by localized renal damage due to reperfusion. The subsequent inflammatory response and leukocyte infiltration can result in extensive tissue damage and, ultimately, acute renal failure. As yet, there is no effective treatment to prevent possible renal damage after ischemia or to control subsequent damage caused by inflammation. The mechanism behind I-R is not fully understood, yet several studies have demonstrated that the ensuing renal damage is mediated in large part by the adhesion of neutrophils and lymphocytes to the vascular endothelium of the graft through ICAM-1 interactions. Two recent studies, using a syngenic renal transplant animal model, demonstrated that antisense targeting of ICAM-1 expression is helpful in preventing I-R injury [8,9]. Both studies employed liposome-complexed phosphorothioate oligomers targeting the 30 end of the ICAM-1 mRNA. One study paired the oligomer with FTY 720 (2-amino-2-[2-(4-octylphenyl) ethyl] propane1,3-diol hydrochloride), an analog of sphingosine which affects the leukocyte response to chemokines, whereas the other examined the influence of the oligomer alone [8]. In both cases, antisense oligonucleotides to ICAM-1 demonstrated a reduction in leukocyte infiltration, as seen by histological examination, and improved renal function compared with animals receiving no treatment or a control oligonucleotide. Furthermore, a mild additive effect was seen when the antisense oligonucleotide was paired with FTY 720 [9]. Although I-R can be induced independently of antigen-specific responses, in the clinic allogenic transplants are likely to be the rule, rather than the exception. For anti-ICAM therapy to be useful, it would need to be effective under these conditions. Application to an allograft was recently shown to work in a Lewis donor into a renal damageresistant AugustXCopenhagen Irish recipient rat model using a methoxyethyl-modified phosphorothioate to ICAM-1 [10]. Both intravenous and oral delivery of the antisense oligonucleotide did extend kidney graft survival, indicating that anti-ICAM-1 could be effective in the clinic. VLA-4
VLA-4 integrin is a heterodimeric molecule, consisting of an a4 and a b1 chain, expressed on most leukocytes and is involved in leukocyte trafficking into a variety of organs. Monoclonal antibodies specific for the a4 chain have proven to be effective in suppressing autoimmune disease in several animal models, and very early results from clinical trials for the treatment of CD and MS held great hope for a successful therapeutic agent. Antisense oligonucleotides to the a4 chain administered as a prophylactic Current Opinion in Pharmacology 2005, 5:508–512
or therapeutic treatment also work well in mouse experimental autoimmune encephalomyelitis (EAE) experiments [5].
Caveats to targeting adhesion molecules Leukocyte adhesion deficiency caused by specific defects in lymphocyte function-associated antigen 1 and Mac1 in humans or knockout mice results in recurrent bacterial infections and impaired wound healing. Even though indications of such complications have not emerged in any ICAM-1 trials, significant side effects have been reported in trials targeting VLA-4 with antibody. The US FDA had granted accelerated approval of the anti-VLA-4 (natalizumab) in November 2004 after reviewing a single year’s worth of data from two MS Phase III studies. Additionally, initial results in CD looked equally promising. However, after only a few months, the manufacturers of natalizumab voluntarily suspended the drug on 28 February 2005, owing to adverse effects that occurred in two patients treated with natalizumb along with interferon b1a, with two confirmed deaths [11]. These adverse events, including the deaths, were found to be due to progressive multifocal leukoencephalopahty (PML). In both cases, indications of a JC virus infection, a causative agent of PML, were observed. It is plausible that reduced immune surveillance brought on by the therapy led to the reactivation of latent virus. In light of these recent events, further development of antisense antiadhesion therapy will have to proceed with caution or be reevaluated.
Soluble mediators of inflammation and receptors Cytokines and chemokines can facilitate the homing and activation of lymphocytes and have a major role in inflammatory disease and graft rejection. Antisense studies targeting expression of these molecules or their cognate receptors have been performed in several animal models. In one study, antisense oligonucleotides to tumor necrosis factor were found to be effective in ameliorating acute and chronic inflammatory bowel disease in two different mouse models [12]. Atopic dermatitis, a human inflammatory disease of the skin, is, in part, caused by the overexpression of interleukin 10 (IL-10) by monocytes and macrophages in the dermis. Anti-IL-10 oligomers delivered by iontophoresis did cause a decrease in IL10 in skin lesions of mice, and a concomitant therapeutic benefit to established dermatitis [13]. Chemokines are the principal factors for directing T cells to allografted tissue. Several different chemokines can bind to the CXCR3 receptor expressed on memory and activated T cells to promote the rejection of skin allografts. Peptide nucleic acid antisense oligomers targeting CXCR3 expression were found to be effective in prolonging the grafts in mice by inhibiting the migration of T cells to the graft [14]. Moreover, this did not interfere with other T cell functions. www.sciencedirect.com
Antisense approaches to immune modulation for transplant and autoimmune diseases Mourich and Marshall
Co-stimulatory molecules and T regulatory cells The goal of an ‘ultimate’ immunosuppressive therapy would be to produce a state of tolerance to transplant tissue antigens or autoantigens. Controlling inflammation might, at best, have only a transient effect and require constant application of the antisense agent. DCs have a key role in initiating some mechanisms of antigen-specific tolerance. Manipulation of DC function (‘engineered DCs’) has been shown to be an effective method for inducing tolerance in autoimmune and transplant models [15]. Phenotypically ‘immature’ DCs express low levels of major histocompatibility complex molecules and costimulatory ligands (CD80, CD86 and CD40). DC maturation is induced by inflammatory signals, resulting in increased expression of these molecules, which are crucial for T cell activation [16,17]. Therapeutic administration of immature DCs can result in deletion of responding T cells and therefore tolerance through nonresponsiveness. A form of active tolerance can also be induced by immature DCs, whereby responding T cells take on a suppressive phenotype. These naturally occurring cells are referred to as regulatory T cells and possess very potent mechanisms for suppressing autoaggressive immune responses [18]. Antisense targeting of co-stimulatory molecules has been applied to both type 1 diabetes mellitus and allograft acceptance. Using a heterotopic heart transplant model in mice and DCs treated with antisense oligonucleotide to CD80 and CD86, it was shown that graft survival could be prolonged through depletion of allospecific T cells [19]. In the NOD mouse model, DCs treated with antisense oligonucleotides to CD40, CD80 and CD86 promoted the expansion of a regulatory T cell population and significantly delayed the onset of diabetes [20]. Co-stimulatory molecule targeting has shown some success when antisense oligonucleotides are administered to the whole animal [21] but only a single injection of antisense-modified DCs was necessary to produce tolerance in the two studies above [19,20], confirming the remarkable utility of ex vivo antisense therapy.
Conclusions It is clear that the development of antisense-based therapeutics is expanding into nearly all areas of human disease. The immune system offers a vast frontier of possibilities for targeted manipulation either to suppress unwanted responses or to augment those that could be beneficial. The inherent specificity of mRNA sequences offers researchers, through antisense, an unambiguous platform for drug design. Molecules with unique roles in specific immune-related diseases are continuously being defined [22,23,24]. As much needed improvements are made to the delivery of antisense oligonucleotides into immune cells, the development of numerous therapeutic agents will certainly follow. www.sciencedirect.com
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Acknowledgements Special thanks to Marta Johnson for her help in gathering literature which has been crucial to the writing of this manuscript.
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