Gene therapy in immune-mediated diseases of the eye

Progress in Retinal and Eye Research 22 (2003) 277–293

Gene therapy in immune-mediated diseases of the eye$ Uwe Pleyera,*, Thomas Ritterb a

Department of Ophthalmology, Humboldt University, Charite Campus Virchow, Augustenburger Platz 1, D-13353 Berlin, Germany b Institute of Medical Immunology, Charit!e Humboldt University, Schumannstrae, D-10098 Berlin, Germany

Abstract Therapy of ocular immune-mediated diseases has changed dramatically over the past two decades. Although a variety of nonspecific immunosuppressive agents are introduced, with advances in cell biology a number of more specific therapeutic options will become available. Gene therapy has the potential to interfere with the immune response at different steps modulating the microenvironment of the eye. In this chapter we focus attention on the most promising candidate genes for gene therapy in ocular immune diseases. Furthermore, we outline the current techniques for delivering genes of interest with their potential merits and drawbacks in the field of ophthalmology. Many of these approaches are still in early phases of study for the treatment of clinical relevant immune-mediated diseases. r 2002 Elsevier Science Ltd. All rights reserved.

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Principles of the immune response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Gene therapy — an introduction . . . . . . . . . . . . . 3.1. Gene transfer with non-viral techniques . . . . . . 3.2. Gene transfer with viral vectors . . . . . . . . . . 3.2.1. Retrovirus . . . . . . . . . . . . . . . . . 3.2.2. Adenoviral vectors . . . . . . . . . . . . . 3.2.3. Adeno-associated virus vectors . . . . . . 3.2.4. Herpes virus ðHSV-1; CMV; EBVÞ vectors .

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Gene therapy in immune-mediated diseases of 4.1. Autoimmune diseases . . . . . . . . . . 4.1.1. Autoimmune uveitis . . . . . . 4.1.2. Dry eye syndrome . . . . . . . 4.2. Transplantation . . . . . . . . . . . . . 4.2.1. Keratoplasty . . . . . . . . . . 4.2.2. Retina transplantation . . . . .

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Infectious diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. HSV keratitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Current problems and further directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Supported in part by Deutsche Forschungsgemeinschaft (Pl 150/10-1 and Ri 764/6-1). *Corresponding author. Tel.: +49-30-450554131; fax: +49-30-450554091. E-mail address: [email protected] (U. Pleyer).

1350-9462/03/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 0 - 9 4 6 2 ( 0 2 ) 0 0 0 4 6 - 0

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1. Introduction It has been recognized for more than 100 years that the eye is an organ prone to immune-mediated diseases. Despite a range of mechanisms that protect the delicate organ against exogenous and endogenous pathogens, inflammatory and immune-mediated processes are a leading cause for blindness (Thylefors et al., 1995). They may affect all compartments and structures of the eye and vary from ‘‘allergic’’ conjunctivitis to infectious associated keratitis and intraocular inflammation. Immunotherapy to affect or modulate the immune system may induce a change in the expression of disease and provide effective strategies for the management of potential blinding diseases. A number of ‘‘classical’’ agents ranging from corticosteroids, immunophilin ligands (Cyclosporin A, Tacrolimus), to cytotoxic agents such as cyclophosphamide have moved to the clinical arena (Sherif and Pleyer, 2002). However, even when enormous progress has been achieved in the field of immunopharmacology, several disadvantages still remain. Currently available immunosuppressive agents have modest selectivity, limited efficacy and still considerable toxicity. Since in almost all immunemediated conditions long-term treatment is required, undesired side effects are a constant treath for the patient and therefore further therapeutic options are necessary. Significant progress has been made in the understanding of basic biology in particular of the immune regulatory network and this may soon turn to medical advantage. A number of new approaches are still in early phases of study for the treatment of clinical relevant immune-mediated diseases. One of the most recent advances in this respect is the introduction of gene therapy. Most of these strategies seek to transfer genes encoding immunomodulatory products that will alter the host immune response in a beneficial manner. By this means gene therapy overcomes obstacles to the targeted delivery of proteins and RNA, and improves their efficacy while providing a longer duration of effect, and, potentially, greater safety. Additional genetic strategies include DNA vaccination and the ablation of selected tissues and cell populations. There is considerable evidence from animal studies that gene therapies work: examples include the treatment of experimental models of rheumatoid arthritis, multiple sclerosis, diabetes, and lupus erythematodus. Up today, more than 400 clinical studies have been performed or are in process (Human Gene Transfer Protocols, 2002; Costa et al., 2000; Seroogy and Fathman, 2000; Evans et al., 2000). However, so far limited experience has gained in respect of gene therapy in ocular diseases, despite the fact that the eye might be even ‘‘an

ideal organ’’ providing several unique advantages including *

* *

*

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a significant number of ocular diseases have been delineated at the molecular level, there are appropiate animal models available, the morphology and function of the eye is relatively easy to evaluate, the ‘‘immune-privileged’’ status of the eye is in advantage to the development of gene therapeutic approaches, as a paired organ the eye holds the opportunity that the second eye can be used as an ‘‘internal control’’.

This chapter is intended not only to provide an overview on the use of gene therapy in the setting of ocular immune-mediated diseases but also to introduce this application in broader respect and to stimulate interest in this field.

2. Principles of the immune response The immune system plays a central role in maintaining homeostasis and health, although it may also cause pathologic reactions to the host in response to exogenous or endogenous pathogens. Immune effector functions can be broadly divided into two types, cell mediated and humoral. Cell-mediated immune responses include the presentation of a pathogen by specialized antigen presenting cells (APC) such as dendritic cells and activation of macrophages with the induction of cytotoxic CD8+ and CD4+ T cells, whereas the humoral immune response is characterized by the differentiation of na.ıve B cells upon antigen contact into antibody secreting plasma cells. Both effector mechanisms activate unspecific inflammatory pathways that eventually eliminate the pathogen. The role of T cells and their activation is one of the critical steps in a number of relevant conditions such as transplant rejection and the induction of autoimmune processes. T cell activation is dependent on two signals: signal 1 is transmitted through the engaged T cell receptor (TCR), whereas the second costimulatory signal is transmitted by the interaction of surface molecules on the T cell (e.g. CD28 and CD40 ligand) and the APC (B7 and CD40) (Fig. 1). Interference at this early step of T cell activation is an interesting approach to abrogate an antigen-specific immune response. In the absence of costimulation, the antigenic signal alone is not sufficient to lead to T cell activation but induces a state of anergy of the engaged T cell. Cytotoxic T lymphocyte associated antigen-4 (CTLA-4), a protein homologous to CD 28, binds B7 with high affinity and interferes with the B7-CD28 interaction (negative signalling). Several studies have shown the

U. Pleyer, T. Ritter / Progress in Retinal and Eye Research 22 (2003) 277–293

279

Induction of immune response Processing

Presentation

Activation IL-2R

B7-1 (CD80) CD28

IL-2

Antigen

APC

TH

MHC II CD4 Peptid

TCR

Fig. 1. Gene therapy to interfere T cell activation. T cells recognize antigens and become activated through interactions between TCR-MHC (signal 1) and co-stimulatory molecules (CD28-B7; CD40L-CD40) (signal 2). Blocking of costimulatory signals leads to T cell tolerance to the presented antigen. Activated T cells secrete cytokines which activate immune cells such as macrophages, B cells, cytotoxic T cells and NK-cells.

beneficial effect of systemic administration of CTLA4Ig, a soluble fusion protein that mimics CD28 in allograft rejection and autoimmune diseases. Gene transfer strategies offer an approach to deliver these proteins to the target organ to modulate the immune response. In case these proteins can be expressed at the site of activation, it offers the advantage of limited toxicity relative to systemic delivery of immunosuppressive treatment. Following T cell activation a delicate network of mediators e.g. cytokines and chemokines is able to boost the immune response and further orchestrate the defense system. Selected cytokines are able to down-regulate the immune response and therefore are also candidates for therapeutic approaches. It has become clear that the T cell mediated-immune response is regulated by two distinct types of CD4+ cells, which secrete different patterns of cytokines that modulate their effector functions. Th1 cells mainly produce pro-inflammatory cytokines (e.g. IFN-g, IL-2) which support cell-mediated immune reactions. In contrast, Th2 cells secrete cytokines (IL-4, IL-10) which are involved in the activation of an humoral immune response. Expression of certain cytokines may shift the local balance between T cell subsets towards an immunosuppressive action. Based on the Th1/Th2 ‘‘paradigm’’ (Mosmann and Coffman 1989; Pleyer et al., 2000a, b) it might be conceivable to modulate allograft rejection by over-expression of Th2 cytokines (IL-4; IL-10). Even when not absolutely conclusive, several studies were able

to demonstrate this effect in solid organ transplant models (Dallmann, 1993; Yamada et al., 1999; Pleyer et al., 2000a, b). In addition, other pathways of elimination of antigen reactive T cells have been investigated. One current approach includes the induction of apoptosis in these cells. Analysis of apoptotic pathways in lymphocytes has shown an essential importance of the family of apoptosis molecules in controlling of the immune system. For example attempts have been made to induce Fas Ligand (FasL) in donor cells or APC (Matsui et al., 1999). However, there are still concerns regarding the proinflammatory effects of FasL recruiting lymphocytes to the target site.

3. Gene therapy — an introduction The tremendous advances in molecular medicine and biology within the last decades have led to the development of promising therapeutic tools for the treatment of cancer, genetic defects, infectious diseases or autoimmune disorders. These include the generation of gene-based approaches, e.g. for the correction of defective genes or the over-expression of therapeutic molecules. Despite enormous efforts and funding which have been put in the development of gene-based therapies the success of these strategies was rather limited. This is mainly due to still unsolved problems concerning the suitable gene transfer vehicle (e.g. the

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viral vector), the efficiency of gene transfer and problems related to immune reactions of the treated patient against the vector or the transferred therapeutic gene. In principle the techniques of introducing genetic material into cells or tissues can be divided into two application forms: gene transfer systems using recombinant viruses as gene transfer vehicle or techniques based on non-viral methods. Non-viral gene transfer include physical and chemical approaches to transfer recombinant DNA into the patient (for review see Nishikawa and Huang, 2001). Viral vectors include different families of viruses which have been turned from an infectious into a therapeutic agent by genetic manipulation e.g. by deletion of viral genes essential for virus replication or assembly (for review see Walther and Stein, 2000; Kay et al., 2001).

to their low efficiency in vivo (Nishikawa and Huang, 2001; Pleyer et al., 2001c). However, gene transfer in the cornea thereby targeting cells of epithelial, stromal or endothelial origin might be a feasible approach. In fact topical administration of plasmid-DNA (IL-4, IL-10 and CTLA4-Ig) on the cornea by gene gun technology . has lead to the prolongation of allograft survival (Konig Merediz et al., 2000). Recently, Oshima et al. have shown successful reporter gene transfer in corneal stroma cells by electric pulse technology (Oshima et al., 1998, 2002). In addition, injection of genetic material in the eye either in its naked form or in conjunction with liposomes (subretinal, intravitreal, subconjunctival) is an interesting option for gene therapy (for review: see Pleyer et al., 2001a, b, c; Pleyer and Dannowski, 2002).

3.1. Gene transfer with non-viral techniques

In contrast to gene transfer technologies using nonviral strategies, viral vectors are generally characterized by their high transduction efficiency of different cell types and tissues, although they do not show the same transduction efficiency of a given cell type (Kay et al., 2001). Therefore, the selection of a gene therapy vector is critically dependent on the cell type or tissue being the target for gene therapy. Toxicity and the induction of an immune response against the vector are still major problems which have to

Several techniques have been developed so far for the transfer of genetic material into cells or tissues without using virus vectors as shuttle. These techniques include electroporation, the use of gene gun, formation of liposomes or simple injection of ‘‘naked’’ DNA (Fig. 2). In contrast to viral vectors, these gene transfer techniques generally do not induce an immune response by the recipient, although their application is limited due

3.2. Gene transfer with viral vectors

Fig. 2. Schematic presentation of liposome mediated gene transfer. The gene is cloned into an expression vector, which is mixed with the liposomes. After packaging of DNA, the lipid/DNA complex attaches to the cell surface, followed by internalization of DNA. If endocytosis is involved, DNA escapes from the endosomes and DNA expression occurs in the cell nucleus.

U. Pleyer, T. Ritter / Progress in Retinal and Eye Research 22 (2003) 277–293

be solved before gene therapy with viral vectors will be considered as therapeutic option e.g. in non-lethal diseases. Different virus families have been investigated for their potential as gene therapy vector (see Table 1). These include vectors derived from: 3.2.1. Retrovirus Oncoretrovirus (e.g. Moloney murine leukemia virus [MoMuLV]. Lentivirus (human/simian immunodeficiency virus [HIV, SIV]). Adenovirus: Adeno-associated virus Herpesvirus (Herpes-Simplex virus type 1, Cytomegalovirus, Epstein-Barr virus) Retroviral vectors: Retroviruses are single stranded RNA viruses surrounded by a lipid envelope. Upon entry into the target cell, the RNA is released, reverse transcribed into double-stranded DNA which is stably integrated into the host DNA. This family of viruses consists of several subgroups which have been investigated — although they may cause human diseases e.g. tumour induction or immunodeficiency (Coffin et al., 2000) — for their potential as gene therapy vector. In contrast to retrovirus vectors based on HIV or SIV genomes (lentiviruses), retroviruses belonging to the subgroup of oncoretroviruses (e.g. MoMuLV) are strictly dependent on cell division and break-down of

281

the nuclear membrane for integration in the cellular DNA. Therefore, these vectors do not seem to be very useful for in vivo transduction of ocular cells since these tissues mainly consist of differentiated, non-dividing cells. For the identification and isolation of ocular stem cells, however, these vectors might be very useful (Bradshaw et al., 1999). The deletion of viral genes (gag, pol, env) in the virus genome allowed the construction of vector genomes harbouring therapeutic DNA up to 8 kb (Kay et al., 2001). Since the generation of infectious recombinant retroviral particles ultimately requires the production of the gag, pol and env proteins, packaging lines have been established stably expressing these essential components for viral replication and assembly (Miller, 2000). This construct design was an important prerequisite in the generation of gene therapy vectors from retroviruses by reducing the possibility of generating a wild type virus since several recombinatorial events would be necessary (Kay et al., 2001) (Fig. 3). Due to the requirements of oncoretrovirus particles with regard to dividing cells, gene therapy trials have been mainly focussed on transduction of cells from the hematopoetic compartment. Recently, the potential of vectors based on HIV or SIV have been investigated for its potential as gene therapeutic vehicle. Due to its natural tropism for CD4+T cells and dendritic cells, lentiviral vectors have been mainly used for gene transfer in hematopoetic cells. However, the possibility of generating packaging lines with other envelope proteins (e.g. the envelope G-protein) from the vesicular stomatitis virus (VSV) has expanded the host range of

Table 1 Vectors for gene transfer Viral

Stable/ Transient

Infection of non-dividing cells

Packaging capacity (kb)

Advantage

Disadvantage

Retrovirus (MoMuLV)

Stable

No

Up to 8

Integration. Many cell types

Instability. Insertional mutagenesis

Lentivirus (HIV)

Stable

Yes

Up to 10

Transduces both quies. and prolif. cells

Safety concerns by HIV-based vectors

Adenovirus

Transient

Yes

Up to 8

Transduces both quies. and prolif. cells. High titer

Immunogenic

Herpes simplex virus

Transient

Yes

> 30!

Tropism for neural cells

Safety concerns

Adeno-associated virus

Stable (?)

Yes

Up to 5

Low immunogenic

Production of high titer stocks

Non-viral

Stable/ transient

Transfection of non-dividing cells

Naked DNA Liposome DNA conjugates

Transient Transient Transient

Yes Yes Yes

Non immunogenic. Repeated Low efficiency. High transduction possible. cell type dependent Transduces both quies. and variability. Toxicity prolif. cells. Easy to handle — — —

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Isolate nucleic acid

Remove essential parts

Virus

Insert therapeutic gene

Vector

Transfer in packaging line Re-assembly of the virus

Fig 3. Strategy for the generation of a gene therapy vector from a wild type virus. As a first step, isolation of the nucleic acid from the wild type virus has to be performed. After the removal of essential virus-specific genes to increase vector safety and space for foreign genes, the therapeutic gene can be inserted into the remaining viral sequences by standard molecular cloning techniques. Then, this construct containing the viral nucleic acid and the therapeutic gene is transfected into a packaging line producing in trans the viral proteins originally deleted from the virus. Alternatively a helper virus can be used to provide viral proteins in trans which are essential for replicating of the viral nucleic acid and for assembly of the vector particles. Gene therapy vectors can be harvested from supernatant of the transfected packaging line or by cell lysis and further prepared for therapeutic gene transfer.

lentiviral vectors. These vehicles efficiently transduce cells of the central nervous system (Naldini et al., 1996). Considering safety problems by using vectors based on HIV several groups have been investigated the potential of gene therapy vectors based on simian immunodeficiency virus (SIV, not pathogenic in humans) which might reduce the risk of these constructs for human gene therapy. 3.2.2. Adenoviral vectors Beside retroviral vectors adenoviruses (Ad) are the most frequently used carriers for the transfer of genetic information in human gene therapy trials. They consist of an icosaedric capsid of 70 nm in diameter harbouring a double-stranded, linear DNA of approx. 36 kb. More than 50 serotypes belonging to different subgroups are known. So far no severe human diseases are known to be associated with adenoviruses, although they may induce tumours in rodents. Interestingly, the eye is a potential target tissue for adenoviruses since infection of the eye may result in a severe keratitis mainly affecting the corneal epithel. However, this disease will not occur with adenoviral gene therapy vectors since essential genes for virus replication are deleted. In addition, it is of interest that gene-modified Ad affects predominantly the cornea endothelium whereas the epithelium is almost not susceptible for Ad-mediated gene transfer.

The deletion of viral genes important for DNAreplication and transcription (E1a, E1b) opened the possibility to use these adenoviruses as vectors for gene therapy (Hitt et al., 1997; Kay et al., 2001). Up to 8 kb of foreign DNA can be efficiently inserted into the adenovirus vector. In addition to its large packaging capacity Ad-vectors have several advantages which make them superior to other gene therapy vectors: (1) they can transduce both dividing and non-dividing cells equally efficient; (2) they can be propagated in vitro at high titers (up to 1011 infectious virions/ml) which is a prerequisite for in vivo experiments and (3) the expression levels of the therapeutic gene are generally very high. On the other side, however, adenovirus vectors induce a cascade of immune reactions against the vector itself and against the transduced cells or tissues which finally leads to transient expression of the desired transgene (Yang et al., 1995; Kay et al., 2001; Ritter et al., 2002). In addition, repeated application of Ad-vectors is difficult to achieve due to the generation of neutralizing antibodies after the first injection of the Advector. Despite the generation of immune responses against Ad-particles, numerous studies have been performed using adenoviral vectors in gene therapy trials mainly in treating end-stage tumour patients (Cao et al., 1998; Roth and Cristiano, 1997; Hermiston, 2000).

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3.2.3. Adeno-associated virus vectors Since it has been realized that the immune responses induced by injection of recombinant adenovirus are a major disadvantage in Ad-mediated gene therapy protocols, other vector systems have been investigated for their potential as gene therapy vector. One promising candidate is the Adeno associated virus (AAV) originally identified as contamination in adenovirus preparations. AAV belongs to the family of parvoviruses and contains single stranded DNA. Six serotypes (AAV 1-6) with different cell specificity have been identified so far without pathologic signs upon infection in humans. The AAV is a small DNA-virus, containing only two viral genes (rep and cap) encoding for proteins essential for virus replication and encapsidation (Muzyczka, 1992; Monahan and Samulski, 2000; Walther and Stein, 2000; Kay et al., 2001). The deletion of the rep/cap genes in the AAV-genome opened its use as gene therapy vector although these genes have to be provided in trans either by a packaging line or by helper virus. In contrast to adenovirus vectors AAV does not induce a strong immune response (no potentially immunogenic viral protein is remaining) making them suitable as gene therapy vector. Although limited packaging capacity is available (4–5 kb) most of the therapeutic genes should be successfully transferred with AAV. Clinical trials using AAV are in progress for the treatment of cystic fibrosis, hemophilia and muscular dystrophy (Wagner et al., 1999; Stedman et al., 2000; Kay et al., 2000). 3.2.4. Herpes virus ðHSV-1; CMV; EBVÞ vectors Recently, the family of herpes viruses (HerpesSimplex virus type 1, Cytomegalo-virus, Epstein-Barr virus) has been target of investigation as a promising candidate for gene therapy vector (for review see: Kay et al., 2001). Upon infection, Herpes viruses persist in humans after primary infection in a latent state for a long period of time without disease making them suitable for long-term expression of therapeutic genes. Vector constructions are in progress, where reactivation and production of infectious virus particles in transduced cells is eliminated. Herpes virus vectors are large viruses containing a significant amount of DNA (e.g. HSV-1 contains 152 kb of DNA). Subsequently many viral proteins are encoded by the HSV-1 DNA. Some of them are not required for virus replication in vitro, making them interesting as gene therapeutic vehicle by insertion of therapeutic genes for deleted non-essential HSV-1 genes. HSV-1 is able to transduce a variety of cell types very efficiently, although due to its persistence in neural cells, most of the studies performed so far tried to target neural cells or tissues (Wolfe et al., 2001). The prevalence of HSV-1 for infecting neural cells makes it very interesting e.g. for the genetic modification of the cornea or the optic nerve. In addition, infection of the eye with HSV may also lead to corneal keratitis

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indicating that herpes virus vectors unable to replicate might be also an interesting tool in the future for targeting corneal cells.

4. Gene therapy in immune-mediated diseases of the eye 4.1. Autoimmune diseases Background. Activation of T cells by self-antigens is under strict control by different mechanisms, both thymic and peripheral. Failure of these mechanisms controlling tolerance to self-antigens may lead to autoimmunity. One of the striking characteristics of autoimmune diseases is the increased frequency of alleles for specific human leukocyte antigens (HLAs) in affected individuals. It seems likely that disease associated HLA molecules have the capacity to bind the antigen and present it to T cells thereby inducing the autoimmune process. Autoimmune diseases are characterized as immune attack on self-tissue. For the eye these include a heterogenous group of extra — as well as introcularly manifesting inflammatory diseases present. ing e.g. as ‘‘dry eye’’ conditions in Sjogrens syndrome, autoimmune-mediated corneal ulcerations, scleritis and autoimmune uveitis. Current therapies for almost all these diseases utilize potent and non-specific immunosuppressive regimens. These therapies are complicated by their side effects and also place the patient at increased risk for opportunistic infections and malignancies. Our current understanding of immune mechanisms underlying most of these diseases remains limited. Although many autoimmune disorders do not have a strong genetic basis, their treatment may nevertheless be improved by gene therapies. Ongoing studies include efforts to identify genes that predispose an individual to develop autoimmunity, identification of autoantigens that trigger or perpetuate autoimmunity, and studies of immune cell interactions that lead to deteriorate the immune response. Pre-clinical success in treating animal models of rheumatoid arthritis has led to the first clinical trials of gene therapy for an autoimmune disease. Since several of the underlying immune mechanisms of autoimmune disorders are redundant and the principles to modulate them are likely to be similar, some basic mechanisms will be discussed. In addition, so far rather limited experience of gene therapy for ocular autoimmune diseases will be focussed on autoimmune . uveitis (AU) and Sjogrens syndrome. 4.1.1. Autoimmune uveitis The underlying immunopathogenic mechanisms of AU are shared with other cell-mediated autoimmune diseases. The initiating of the disease by organ-specific antigens such as retinal S antigen and IRBP and the presence of T cells in uveal tissue at onset of the disease

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is well documented. Importantly, established animal models exist that serve as models for human autoimmune uveitis and have permitted insights into the pathophysiology and new treatment options. To modulate uveitis by gene therapeutic approaches there are several options that can be grouped in two broad categories. The first is preventive therapy in genetically predisposed individuals whereas the second approach is instituted in patients that are already suffering from uveitis. Prevention of autoimmune uveitis. In principle the best approach for AU would consist of preventing induction of intraocular inflammation. This would include identifiying people at risk developing the disease depending on highly reliable methods for ‘‘pre-uveitic’’ people and a better understanding of the underlying mechanisms. So far the etiology of AU is still not known. Even when there exist some indications that several subclasses of AU are associated with an underlying HLA subtype that predispose for e.g. VKH (HLA-A29) or Behcets disease (Mitzuki et al., 1992), no single mutant gene is known whose replacement with gene therapy would reverse the course of the disease. Thus, in vivo gene therapy with predisposing genes is yet not a realistic prospect before improvements have been made in the early diagnosis of uveitis. Gene therapy in ‘‘primed’’ individuals. One of the challenges in the clinical setting is that the uveitis patient

presents an immunologically primed state and may even recruit new pathogneic clones in the presence of chronic recurrent uveitis. There are several approaches conceivable to prevent and modulate AU using gene therapy (Fig. 4). 4.1.1.1. Tolerance induction. The aim to prevent immune-mediated damage might be reached considering approaches to induce central and peripheral tolerance. Whereas no documented attempt exists to induce central tolerance in uveitis, successful trials have been reported in other autoimmune diseases. Intrathymic injection of islet extracts resulted in the deletion of islet reactive thymocytes and suppressed type 1 diabetes onset (Posselt, 1992). Additional studies suggested that intrathymic gene delivery using adenoviral vectors resulted in tolerance induction to the virus encoded antigen (DeMatteo et al., 1995). The feasibility and safety of intrathymic injection of donor leukocytes in a clinical setting could be demonstrated in heart transplant recipients (Remuzzi et al., 1995). Taken together these results are encoraging, however it has to be kept in mind that intrathymic induction of tolerance may differ in the primed postpubescent individual. 4.1.1.2. Modification of antigen presentation. It is generally recognized that autoimmune disease induction occurs due to a breakdown of protective tolerogenic

Targets for intervention in autoimmune diseases Inhibition of CD4-MHC II interaction

Antigen presenting cell

MHC blockade

Autoreactive T cell

Tolerance induction

T-cell receptor antagonism Fig. 4. Targets for intervention in autoimmune diseases. T cell activation can be inhibited by blocking the MHC binding site; blockade of the CD4 molecule expressed on T cells and T cell depletion by specific monoclonal antibodies. These strategies are rather non-selective since only a fraction of the peripheral T cell repertoire is inhibited. More specific modulation is attempted by inducing selective T cell anergy by administration of autoantigenic peptides in tolerogenic form; or autoantigen analogues acting as TCR antagonists. Alternatively, enhancing regulatory T cell might be induced by vaccination like treatments to control the activity of autoreactive T cells.

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mechanisms that control self-antigens before these are recognized as non-self and become the target of an immune response. The early step of breakdown requires antigen uptake and processing by APC with subsequent presentation to CD4+ cells in the context of proinflammatory cytokines. A number of factors are responsible that may lead to this change including genetic predisposition, antigen dose, cytokine microenvironment and TCR ‘‘promiscuity’’. Therefore, APCs including resident dendritic cells and macrophages are possible targets for gene delivery. Potential ways to interfere with induction of autoimmune diseases include gene transfer of molecules that prevent APC activation, prevent processing of antigen, prevent upregulation of adhesion molecules or promote APC-responsive lymphocyte death. Dendritic cells. Dendritic cells (DC) are the most potent APC that respond to local injury by uptake of (soluble) antigens, processing and migration to local lymphoid tissue. Activated DC express adhesion molecules that enable them to interact with endothelial receptors and subsequently recruiting T cells. Therefore, one strategy to interfere with antigen presentation could be to express soluble cell adhesion molecules (such as ICAM-1, ICAM-3) within the target tissue. Indeed, in some models of autoimmune diseases this strategy has been proven to function and prevented e.g. type I diabetes in NOD mice (Martin et al., 1998). In addition, DC have also the potential to induce tolerance under defined conditions (Steptoe and Thomson, 1996). Although still not completely understood it has been shown that DC are able to suppress T cell activation in autoimmunity as well as in allogeneic immune response. Conditions that favour induction of tolerogeneic DC include exposure to Th2 cytokines such as IL-4, IL-10 or TGF-b. The processes thought to be involved are probably a shift to a Th2-mediated immune response, induction of apoptosis or regulatory T cells. Dendritic cells that have been transfected with genes encoding IL-10, CTLA4-Ig or TGF-b were able to significantly prolong allogeneic graft survival (Takayama et al., 1998, 2000, 2002). Modified DC expressing vIL-10 produced high levels of this cytokine in vitro with subsequent marked reduction of MHC antigen expression resulting in decreased T cell stimulation and induction of T cell hyporesponsivness (Takayama et al., 1998). Interestingly the route of administration of tolerogenic DC seems to play an important role. Whereas intravenous injection of DC result in splenic accumulation, subcutaneous injection of labelled DC demonstrated delivery predominantly to draining lymph nodes (Eggert et al., 1999). It is conceivable that for the purpose of tolerance induction by modified DC the proper local environment and the route of administration are critical prepositions for effective immunomodulation.

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Macrophages. Macrophages have various important functions not only as primary effector cells but also to trigger the immune responses by presentation of antigen to antigen-specific T cells. One of the earliest events in macrophage activation is the production of IL-1. When target cells become exposed to this cytokine they may become sensitized to Fas triggered apoptosis. (Stassi et al., 1997). A series of studies in autoimmune animal models e.g. rheumatoid arthritis or insulitis have shown that gene transfer of IL-1 receptor antagonist protein or TNF receptor can prevent resident APC activation (Le et al., 1997; Ghivizzani et al., 1998). Another potential strategy to interfere at the level of antigen presentation and macrophage activation is related to the c-rel family of transcription factors, including nuclear factor NF-kB. Since expression of a large number of cytokines and adhesion molecules are under control of NF-kB, it is an intriguing approach to inhibit activation of macrophages at this level. Potential ways to interfere are gene transfer of the regulator of NF-kB, IkB which is a physiological inhibitor that is intracellularly bound to NF-kB. Proof of principle has been shown in a number of studies preventing inflammatory response by macrophages and monocytes (Baldwin, 1996, Makarov et al., 1997). 4.1.1.3. Intervention based on costimulatory molecules. Activation of CD4+T cells requires in addition to the MHC class II/TCR complex a second signal that acts through the CD28 molecule at the T cell. Blockade of this interaction leads to T cell anergy or apoptosis. Intervention at the level of costimulatory molecules was achieved by the construction of a fusion protein, CTLA4-Ig that effectively prevents the B7-CD28 interaction. Gene transfer of this molecule was performed in different models and in particular in allo-transplantation models significant prolongation of graft survival was achieved (for review: Najafian and Sayegh, 2000). As already reported, bioballistic delivery of CTLA4-Ig resulted in prolonged corneal graft survival in an experimental model. Moreover, CTLA4-Ig protein or cDNA transfer in the cornea prevented allograft rejection (Comer et al., 2002). 4.1.1.4. Induction of apoptosis. One of the challenges in the clinical setting of an immunological primed uveitis patient is that he may even recruit new pathogenic clones in the presence of chronic recurrent uveitis. To test the possibility that epitope-specific protection can be achieved not only in naive but also in already primed individuals an immunoglobulin — antigen fusion construct was used in EAU. A chimeric retrovirus encoding a major pathogenic epitope of mouse interphotoreceptor retinoid- binding protein (IRBP) in frame with mouse IgG1 heavy chain was transferred into mice sensitized with a uveitiogenic regimen (Agarwal et al., 2000).

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Using this construct peripheral B cells were transduced and subsequently infused into syngeneic recipients. Protection from EAU was achieved not only when treatment was applied before antigen challenge but also when given even 7 days after uveitiogenic immunization. Protected animals had reduced antigen-specific immune response without clear evidence of Th1/Th2 modulation. Antigen-specific protection was not transferable indicating that regulatory T cells did not play a major role. There are several points that need to be emphasized from these results: first there is a clear advantage of using ex vivo transduced autologous cells over direct application of the retrovirus since it avoids the risk of secondary immune response directed against the vector. This is particular important considering that repeated applications might be necessary. Secondly, since the autoimmune mechanisms are essential similar for other tissue-specific autoimmune diseases, e.g. autoimmune encephalitis or arthritis, this approach is applicable also for these entities. The observation that protection could be achieved also in primed recipients holds promise for further investigations. Still the mechanisms of action are not completely clear in this experiment. There are at least 2 mechanisms that may explain tolerance induction: active suppression by regulatory cytokines or induction of anergy resulting from antigen presentation by B cells in the absence of costimulation. 4.1.2. Dry eye syndrome Like autoimmune uveitis, certain forms of the dry eye syndrome are considered as an autoimmune-mediated phenomenon. Innumerable people throughout the world suffer from dry eyes, which has been considered as one of the major ocular problems in the developed world. . Sjogren syndrome as the severe form of dry eye syndrome is classically defined as the presence of at least two components of a clinical triad consisting of dry mouth, dry eyes and autoimmune disease predominantly rheumatoid arthritis (Fox et al., 1986; Pflugfelder et al., 1996). Clinical diagnostic criteria have been proposed by Fox and colleagues (1986): Abnormally low Schirmer test; presence of rose bengal staining, decrease of salivary gland flow, proof of lymphocytic infiltration in salivary glands, manifestation of systemic autoimmune problems (Fox et al., 1986; Pflugfelder et al., 1996). Several risk factors including genetic, environmental, endocrine and immunologic parameters may play a role in the onset of disease. It has been shown that the lacrimal glands of patients with dry eye show infiltration of inflammatory cells. These cells produce mediators which might be toxic for the lacrimal gland cells leading to dysfunction in tear production. As considered to be important mediators in other autoimmune diseases inflammatory cytokines are suspected . to play an important role for Sjogren’s syndrome as

well. Consequently the repertoire of therapeutic molecules discussed in other autoimmune diseases might be an alternative to conventional treatment of severe forms of dry eye syndromes, however, so far, few experimental studies have been performed. Targeting TNF-a as a key player in inflammation is an interesting therapeutic option. It has been shown that lacrimal cells genetically engineered by Ad-mediated gene transfer to express a soluble TNFRp55-Ig fusion protein (AdTNFRp55-Ig) (Kolls et al., 1994) efficiently inhibit the proliferation of lymphocytes in an autologous mixed cell reaction, an apparent in vitro model of autoimmune dacryoadenitis. In addition, lacrimal gland cells secreting IL-10 were also able to suppress the proliferation of autologous lymphocytes (Zhu et al., 2002). These results were encouraging and have to be proven in in vivo models for their therapeutic potential. First in vivo results indicate that AdTNFRp55-Ig expression suppresses lacrimal gland immunopathology and ocular surface disease in a rabbit model of autoimmune dacryoadenitis (Zhu et al., submitted). For these experiments peripheral blood mononuclear cells were co-cultured with autologous acinar cells for 5 days and co-injected together with the Ad-construct expressing the soluble TNFRp55Ig chimeric construct. In addition, induction of apoptosis of lacrimal gland cells was diminished when the TNFRp55-Ig construct was co-injected. Similar results have been observed when an adenovirus construct encoding for viral IL-10 is being used (Trousdale et al., in preparation). Further gene transfer studies have to prove its functionality and therapeutic value/effect in experimental autoimmune models. 4.2. Transplantation The induction of tolerance against allogeneic transplants (cells, tissues, organs) is the ultimate goal in transplantation biology. Without further treatment allogeneic grafts generally will be rejected within days or few weeks. Immune cells of the transplant recipient are critically involved in this rejection process. Current immunosuppressive treatment protocols to prevent graft rejection have to be administered for a prolonged even lifetime period and may lead to severe side effects including the reactivation of latent virus infections, neoplasia and multiple toxic problems (Tolkoff-Rubin and Rubin 1998). Therefore the genetic modification of the graft prior to transplantation seems to be an attractive approach to protect the graft from allogeneic rejection. A major advantage of gene therapy in transplantation is based on the fact that the transplant can be genetically modified ex vivo, thereby reducing side effects of systemic application of the gene therapy vector in other models. Several strategies to protect the graft from rejection have been investigated including gene transfer of anti-inflammatory soluble cytokine

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receptors (IL-1RA, TNF-R), graft-protective genes (heat shock proteins, anti-apoptotic genes), immunomodulatory cytokines (IL-10, IL-12p40, IL-4) or blockade of co-stimulation (CTLA4-Ig). Some of these applications will be outlined more detailed. 4.2.1. Keratoplasty With more than 60,000 procedures a year, penetrating keratoplasty is easily the most frequent transplantation procedure of human tissue in Europe and the United States (EBAA, 1998). Reports on the incidence of graft rejection after penetrating keratoplasty vary between 5% and 40% (Sanfilippo et al., 1986; Pleyer et al., 1992; Reinhard et al., 1997). In the presence of risk factors the 5-year prognosis for penetrating keratoplasty is even inferior and estimated to be approximately 50% which is poorer than that for transplantation of parenchymatous organs (Sanfilippo et al., 1986). Continued preventive and therapeutic efforts are therefore required to improve the prognosis after keratoplasty. Immunosuppression can be induced by systemic administration of pharmacological agents. Although Cyclosporin A and corticosteroids have reduced the rejection rate of corneal allografts, still a number of grafts will undergo rejection and the prolonged use of these agents can produce significant side effects that may limit their use in a non life-threatening indication. Therefore, less toxic alternative approaches are needed for corneal transplants. As an interesting approach, genes expressing immunomodulatory proteins can be introduced into corneal cells. These gene-based therapeutic strategies have been suggested as an attractive option in the prevention of corneal allograft rejection (Larkin et al., 1996; Ritter et al., 1999; Pleyer et al., 2000a, b). Indeed, the cornea provides a number of exceptions from other organs that may make it prone to study the effects of this approach. In contrast to other tissues that are routinely transplanted, the cornea can be cultured for a prolonged period of time and therefore allows ex vivo manipulation. In addition, the cornea is easy accessible and its perfect transparency allows direct visual observation. An effective gene transfer particular to corneal endothelial cells, the main target of allograft rejection following penetrating keratoplasty has been previously reported (Larkin et al., 1996; Ritter et al., 1999; Oral et al., 1997). 4.2.1.1. Modulation of Th1/Th2 immune response. The precise mechanisms by which corneal grafts are rejected remain a subject of investigation, but the central role of T lymphocytes is unquestioned. A number of studies indicate that rejection of orthotopic corneal allografts is mediated predominantly by CD4+ T lymphocytes (He et al., 1991; Pleyer et al., 1995). Recently a predominant Th1 type immune response was identified based on the distinct cytokine profile detected within rejected corneal

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grafts as well as in aqueous humor of experimental animals undergoing transplant failure (Torres et al., 1996; Sano et al., 1998; Yamada et al., 1999). Since Th2 cells can down-regulate Th1 cells and biasing the recipient’s immune response in a Th2 direction, it may have a positive effect on corneal graft outcome. IL-4 is regarded as one of the key cytokines that support the Th2 response. Expression of IL-4 is associated with a Th2 response that supports humoral immunity and down-regulation of the inflammatory actions of Th1 cells. In vivo administration of IL-4 has been successfully used in experimental models of autoimmune diseases and in organ transplantation (Takeuchi et al., 1992, 1997; He et al., 1998; Kato et al., 2000). In addition, IL-4 demonstrated another biological effect that may modulate the outcome of corneal graft survival. IL-4 blocked the induction of corneal angiogenesis induced by basic fibroblast growth factor. Detailed studies disclosed an inhibitory effect on the migration of vascular endothelial cells (Volpert et al., 1998). Interleukin-10, originally described as cytokine synthesis inhibitory factor is a potent immunomodulatory cytokine that down-regulates antigen-presentation by APC. In addition, IL-10 inhibits production of monokines such as IL-1, IL-6, IL-8 and TNF-a (De Waal Malefyt et al., 1991a, b; Cassatella et al., 1993; De Vries, 1995). Gene transfer leading to IL-10 expression in allografts may therefore markedly impair effective antigen presentation, reduce graft immunogenicity and inhibit inflammation. In contrast to cellular IL-10 (cIL10), the Epstein-Barr-Virus (EBV) encoded IL-10 homologue (vIL-10) only shows these immunosuppressive properties but not some stimulatory effects on natural killer (NK) cells and cytotoxic T cells (Qin et al., 1997). It has been shown that gene transfer of vIL-10 leads to prolonged survival of cardiac allografts even in a highly histoincompatible strain combination (Zuo et al., 2001). In cornea transplantation, it has been shown that ex vivo transduction of corneas prior to transplantation with an adenovirus encoding for IL-10 lead to prolonged survival in a sheep keratoplasty model (Klebe et al., 2001). However, contradictory results have been observed in a mouse (subconjunctival or intraperitoneal injection of IL-10 protein) and rat cornea transplant model (ex vivo transduction with adenovirus encoding IL-10 (Torres et al., 1999; Sedlakova et al., in preparation). Therefore the role of IL-10 has to be studied in more detail. 4.2.1.2. Intervention based on costimulatory molecules. The inhibition of costimulatory pathways important for full activation of T cells has been shown to be a very successful approach in preventing rejection of solid organ transplants (for review see Najafian and Sayegh, 2000) (Table 2). Consequently, the blockade of

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Table 2 Target molecules for prevention of allograft rejection Effector mechanisms

Potential modulator

Antigen presentation IL-1/IL-1R TNF-a, TNFR-1 Neutrophile activation

soluble (s) IL1-IR-Ig sIL-1-IIR-Ig sTNFR-1Ig sICAM-1, sICAM-2, sCD18 soluble immunoglobulin molecules, e.g.: sICAM-1, sICAM-2, sCD48, sVCAM sFasIg Bcl-2 MHC class I peptide sCR1-receptor, DAF, MCP-1

Adhesion molecules

Fas/FasL Apoptose induction NK cells Complement-mediated destruction

CD28-CD80-CD86 interaction by the application of a chimeric CTLA4-Ig fusion product either as protein or as adenovirus construct has been investigated in experimental keratoplasty. It could be shown that ex vivo treatment of corneas with CTLA4-Ig prolonged graft survival although systemic application resulted in sustained acceptance of allografts (Konig Merediz et al., 2000; Comer et al., 2002). However, systemic immunosuppression mediated by CTLA4-Ig might not only inhibit immune responses against allo-antigens but as well against potential pathogens and should therefore not applied. The inhibition of pro-inflammatory cytokines e.g. TNF-a by the ex vivo modification of corneas with an Ad-construct encoding for a soluble TNF-a binding protein does not seem to prevent graft rejection (Rayner et al., 2001). 4.2.2. Retina transplantation The photoreceptors are a most important part of the retina and ultimatively required for perfect vision. Loss of photoreceptors by various diseases impairs vision which substantially reduces the quality of life. A number of inherited diseases are affecting the integrity of the retina including age-related macula degeneration (AMD) and retinitis pigmentosa (for review see: Bennett and Maguire, 2000; Lund et al., 2001). As a choice of treatment, transplantation of photoreceptors or retina transplants have been considered as a therapeutic option. Although the eye is described to be an immunologically privileged site (ACAID), rejection of retinal allotransplants is frequently observed. Similar to the therapeutic options already described for corneal transplants, genetic modification of retinal cells or transplants might be an useful approach. In principle there are two options of treatment: either the genetic manipulation of the retinal cells or tissues ex vivo or the direct injection of the gene therapy vector in the eye during or shortly after transplantation. So far, gene

therapy studies included transfer of growth factors into retinal cells (e.g. nerve growth factor, BNDF, ciliary neurotrophic factor, neurotrophins, basic fibroblast growth factor (Bennett and Maguire, 2000; Lund et al., 2001). It has been shown that Ad-mediated gene transfer of CNTF leads to reduced loss of photoreceptors both in the retinal degenerate (rd) (Cayouette and Gravel, 1997) and in the retinal degenerate slow (rds) mouse (Cayouette et al., 1998). The injection of an adenovirus encoding for bFGF has improved the survival of photoreceptors in the RCS rat (Akimoto et al., 1999). The introduction of immunomodulatory genes — as already described for cornea transplantation — should contribute to prevent rejection of allogeneic transplants. However — to our knowledge — neither experimental nor clinical studies have been undertaken to investigate the application of immunomodulatory gene therapy as therapeutic option in photoreceptor or retina tissue transplantation.

5. Infectious diseases Despite the success in developing antiviral treatment, gene therapy is considered as an alternative approach particular in patients non-responding to conventional drug therapy. According to Bunnell and Morgan, gene therapy of infectious diseases can be divided into three broad categories: (1) gene therapy based on nucleic acids (e.g. antisense DNA/RNA, RNA-decoys, ribozymes); (2) protein-based approaches (e.g. expression of antiinfectious cellular proteins or intrabodies); (3) immunotherapeutic approaches (e.g. vaccination, cytotoxic T cells or dendritic cells) (Bunnell and Morgan, 1998). To date most of the gene therapy trials in infectious diseases were applied for the treatment of infection with human immunodeficiency virus (HIV) (Human Gene Transfer Protocols, 2002).

5.1. HSV keratitis With regard to ocular virus infections Herpes virus type 1 (HSV-1) infections play a dominant role. HSV-1 is a very common pathogen affecting 60–90% of the adult population. Infection with HSV-1 leads to humoral (antibodies) and cell-mediated (T-helper, cytotoxic, memory) immune response. This prevents the organism from virus-induced morbidity or even mortality. However, infection with HSV-1 is latent and reactivation may lead to the onset of the disease despite or even because of the presence of B- and T-cells specific for HSV-1. HSV-1 is one of the leading causes of

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infectious corneal blindness in the world (Carr et al., 2001). Current antiviral protocols lead to a reduction of ocular infection, however, recurrences are frequently observed. Therefore, HSV-1 is an interesting target for gene therapy. The suppression of potential deleterious immune responses against HSV-1 using anti-inflammatory cytokines might be a promising approach. It is known from previous studies that in animals with spontaneous regression of HSV-1 lesions elevated levels of immunosuppressive cytokines, e.g. IL-10 could be detected (Babu et al., 1995). As a major immunomodulatory cytokine, Interleukin-10 gene transfer is a candidate molecule for the treatment of HSV-1 mediated immune disorders. It has been shown in a mouse model of HSV-1 induced disease that single plasmid transfer encoding for IL-10 lead to a reduction of ocular pathology (Daheshia et al., 1997). Other experiments investigated the potential of reducing the virus load within ocular tissue. Consequently, type I interferons have been analysed for their potential in inhibiting virus replication during ocular HSV-1 infection. It could be shown that IFN-a-gene transfer lead to a reduction of both virus and immune cell infiltration (Noisakran et al., 1999; Noisakran and Carr 2000a, b). However, gene therapy seems to be only successful, when the therapeutic plasmid was applied 24 h before onset of infection (Bunnell and Morgan, 2001). Further studies are necessary to identify successful gene

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therapeutic strategies with the potential to fight HSV-1 mediated diseases when it is already in progress.

6. Current problems and further directions As already indicated, the induction of an antiviral immune response is still one of the major unsolved problems in gene therapy. These immune responses include both cells from the innate immune system (e.g. dendritic cells, macrophages as early defense strategy by the host) as well as components from the aquired immune system (B cells, T cells) (Bromberg and Debruyne, 1998; Brenner, 1999; Kay et al., 2001; Ritter et al., 2002) (Fig. 5). However, there is a clear hierarchy in the severity of introduction of immunity against gene therapy vectors (adenovirus >retrovirus >AAV). The generation of an immune response both against the vector and the transduced cell is problematic since it leads to both reduced and shortened expression of the therapeutic gene (Reichel et al., 1998a, b) and in addition may prevent repeated application of the same gene therapy vector. As an therapeutic option, the application of immunomodulatory genes may reduce the immunogenicity and prolong expression of the therapeutic gene. In fact, it has been shown that Ad-mediated gene transfer of viral IL-10 allogeneic heart transplants inhibits the immune response to both alloantigen and

Three ways how the immune system prevents the success of gene therapy

1

2

APC

Induction of antivector CD4/CD8+ T cells

TH0 B ?

Generation of neutralizing antibodies

3

Target TGF-ß cell

Generation of cytotoxic CD8+ T cells

Fig. 5. Three ways how the immune system prevents the success of gene therapy. (1) Viral particles are taken up by antigen-presenting cells (APC), capsid proteins are degraded and presented via MHC class I pathway to activate virus-specific CD4/CD8+T cells. (2) Neutralizing antibodies eliminate viral particles. (3) Vector-transduced target cells present viral peptides from remaining virus-specific genes after degradation by the proteasome via MHC class I pathway to cytotoxic CD8+T cells.

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adenoviral antigen (Qin et al., 1997). Similar findings have been reported by Reichel and colleagues when they co-injected an Ad expressing CTLA4-Ig in the subretinal space together with an reporter construct. This treatment led to prolonged expression of the reporter gene in retinal cells (Ali et al., 1998). We could show that Ad-mediated gene transfer of a soluble TNF-receptor (AdTNFRp55-Ig) together with an adenoviral reporter construct prevented down-regulation of reporter gene expression and significantly prevented the infiltration of inflammatory cells (T-cells, macrophages, NK cells, granulocytes) in a syngeneic rat heart transplant model (Ritter et al., 2000). Taken together, gene therapy is still in its infancy and even when significant progress has been made in recent years, many issues need to be resolved. These include (1) the choice of a suitable vector with low or no immunogenicity, (2) conditions, which lead to efficient transduction of the target cell or tissue (‘‘targeting’’) only and no spread of the vector in other tissues, (3) expression of the therapeutic gene only under specific conditions (‘‘regulatable gene expression by inducible promoters’’) and (4) efficient and low-budget production of the gene therapy vehicle. In addition, the targets for intervention also need to be more clearly defined and may vary depending on the underlying disorder. In this respect, immunoregulatory cytokines are at the forefront of applications for local immunosuppression, but also other immunoregulatory molecules need to be included in future approaches. Potential targets could include modifiers of cell adhesion molecules as well as modulators of APC. If these problems will be solved, gene therapy of various diseases might be an efficient alternative to conventional drug therapy even in nonlethal applications. This might be particularly true for the eye as an ideal target organ for gene therapy.

Acknowledgements The authors like to thank Drs. Mel Trousdale, M.D., Doheny Eye Institute, Los Angeles, CA, USA, and Hans-Dieter Volk, M.D. Institute of Medical Immunology, Charite! , Humboldt-University Berlin, Germany, for critically reading this manuscript. This work was supported in part by a grant of Deutsche Forschungsgemeinschaft (DFG Pl 150/10-2) and (DFG Ri 764/6-1).

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