Gene therapy in transplantation

Available online at www.sciencedirect.com

Transplantation Reviews 23 (2009) 159 – 170 www.elsevier.com/locate/trre

Gene therapy in transplantation Jerome M. Laurencea , Richard D.M. Allena , Geoffrey W. McCaughanb , Grant J. Loganc , Ian E. Alexanderc,d , G. Alex Bishopa,b , Alexandra F. Sharlanda,⁎ a

Collaborative Transplantation Research Group, Bosch Insitute, Royal Prince Alfred Hospital and University of Sydney, 2006, Australia b AW Morrow Liver Immunobiology Laboratory, Centenary Institute, Sydney, 2050, Australia c Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, 2145, Australia d Discipline of Paediatrics and Child Health, University of Sydney, 2006, Australia

Abstract Gene therapy is an exciting and novel technology that offers the prospect of improving transplant outcomes beyond those achievable with current clinical protocols. This review explores both the candidate genes and ways in which they have been deployed to overcome both immune and non-immune barriers to transplantation success in experimental models. Finally, the major obstacles to implementing gene therapy in the clinic are considered. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Pharmacologic therapies attempt to correct the disease phenotype, leaving the genotype unaltered. Gene therapy is the delivery of genetic material to a target cell or tissue with the intention of ameliorating disease, seeking to alter the disease-associated genotype. Gene therapy can take various forms. Gene transfer provides the cell with an expressible cloned DNA sequence encoding a functional protein intended to achieve a therapeutic effect. A diseased phenotype might also be improved by reducing the production of certain proteins, a process termed silencing. Gene repair is a process directed at correcting a genetic defect in situ and allowing the corrected gene to be regulated endogenously. Although offering great potential for novel genetic manipulation, gene repair and gene silencing technologies are still in their infancy. This review will therefore focus on gene transfer, a process which requires a safe and efficient means of gene delivery. The characteristics of an ideal gene delivery vector are summarized in Table 1. No single gene delivery system possesses all of these characteristics. Each system will possess a group of properties that dictates its suitability for ⁎ Corresponding author. University of Sydney, NSW 2006, Australia. Tel.: +61 2 9351 2897; fax: +61 2 9036 7083. E-mail address: [email protected] (A.F. Sharland). 0955-470X/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.trre.2009.04.001

a particular application. Gene transfer technologies, mainly based on viral vectors, are already being used with some success in human therapeutic trials involving cellular autotransplantation [1,2]. However, this review will concentrate on the application of gene delivery technology in solid organ transplantation where, despite extensive experience in small animal studies, no human trials have been conducted. 1.1. Gene therapy in transplantation Transplantation is already a successful treatment of endstage organ failure [3,4]. Acute allograft rejection is effectively prevented by current pharmacotherapies, but half of all grafts are lost by 10 years after transplantation, and long-term treatment is associated with risks of malignancy, vascular disease, and diabetes [5-8]. The major challenges facing patients with organ failure are a shortage of donor organs and, once transplanted, chronic allograft rejection and complications of treatment of rejection. The most advanced efforts toward gene therapy have focused on replacing a deficient gene in the context of a monogenetic disorder [1,2,9,10]. In the context of transplantation, gene therapy offers the prospect of targeted, life-long therapy to prevent rejection. In addition, the modification of organs from other species to render them transplantable into humans and increase access to trans-

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Table 1 The desirable characteristics of a gene therapy vector Characteristic

Comment

Ease of production Transgene expression profile appropriate to application

High titer preparations should be easy and inexpensive to produce. Target gene expression may need to be sustained (such as when treating phenylketonuria) or may need to be regulated (insulin expression in treatment of diabetes mellitus). An immune response to a vector or transgene may limit the period of effective expression or may promote an immune response to the tissue that expresses it. If the vector delivers the target gene into a specific tissue or cell type, its therapeutic potential may be maximized. The packaging capacity of a vector in terms of size of transgene should not limit therapeutic potential. Ideally dividing cells should continue to express the therapeutic product either by chromosomal integration or formation of an episome that replicates and segregates with cell division. The vector should be able to target cells that are not actively dividing. Host chromosome integration engenders the risk of insertional mutagenesis. This is reduced significantly if integration occurs in specific sites away from critical genes and their regulatory elements.

Mild or no immune response Tissue specificity Vector capacity Continued expression with cell division Ability to target resting as well as dividing cells Site-specific integration

The desirable characteristics of a gene therapy vector [22].

plantation is an important goal. By expressing a therapeutic factor only in the place where it is required, gene therapy may be able to eliminate deleterious effects associated with systemic drug administration. A single dose of a therapeutic gene would be preferable to current pharmacologic therapies, which must be taken regularly and for the life of the graft. Gene therapy also exhibits the potential for the induction of durable, drug-free, immunologic tolerance. Nevertheless, there are many potential problems associated with this modality of treatment, including difficulty regulating transgene expression, safety concerns about malignancy, infection, and inflammation. The application of gene therapy to transplantation only has merit if it can enhance the quantity and quality of life for people with endstage organ failure. 1.2. Modes of gene transfer Genes may be delivered by in vivo gene transfer, where the vector is applied either systemically or locally to the target tissue within the prospective donor or recipient. Alternatively, organs or cells may be collected and modified outside the ultimate host and then returned in a process termed ex vivo gene transfer. Ex vivo transfer limits the exposure of other organs to potentially toxic effects of the vector system, whereas cells modified ex vivo can be selected from those that have failed to undergo modification or expanded in number to allow delivery of a greater inoculum of modified cells. In the realm of solid organ transplantation, whole organs are uniquely accessible for modification ex vivo. However, gene transfer in vivo may be more efficient than ex vivo modification of an organ that is stored cold before transplantation [11]. 1.3. Types of gene delivery vector In general, transfer of DNA into mammalian cells can be readily achieved by a variety of physical (such as pressure or injection), chemical (such as calcium phosphate), or

electrical methods [12,13], collectively known as transfection. Transfection can also be achieved using complexes of nucleic acid and cationic polymers (such as polyamines) known as polyplex systems [14]. Lipid based gene delivery systems include lipid encapsidation of a DNA construct (liposome), cationic lipid and nucleic acid complexes (lipoplexes) [15], and lipopolyplex hybrids of polycationic polymers and lipid [16]. Receptor-mediated uptake of nonviral DNA vectors can be achieved by binding to specific receptors via the conjugation of specific ligands usually to the free amino groups of polycations in the vector and has been shown to increase the efficiency of gene delivery both in vivo and in vitro [17]. The transfer of genetic material using recombinant viruses is known as transduction. Recombinant viral vectors achieve more efficient delivery of nucleic acid than nonviral vectors on a “per molecule” basis [18-23]. Transduction is fundamentally different from infection by a wild-type virus. Transduction is a nonreplicative process leading to the introduction of genetic material into the target cell by the recombinant vector. The only wild-type viral sequences necessarily retained in the recombinant vector genome are those required for genome replication and packaging during vector production and for desirable aspects of the parent viruses' biology. Such sequences are referred to as cis-acting because they regulate the expression of genes on the same nucleic acid strand. Most, or all coding (trans-acting) sequences, are removed. In designing a vector, the therapeutic gene cassette replaces trans-acting elements of the viral genome while maintaining the cis-acting elements (Fig. 1). This generates a vector incapable of replication. The viral coding sequences required for the assembly of viral particles in the packaging cells are provided in trans expressed from heterologous plasmids or incorporated into the producer cell genome. The most commonly used viral vectors in the gene therapy field are derived from adenoviruses (AdVs), gammaretroviruses (RVs), lentiviruses (LVs), and adeno-associated viruses (AAVs). Each system

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Fig. 1. Converting a wild-type virus into a vector. A, The genome of the virus includes the genes required for replication (blue), viral structural proteins (red), and genes involved in viral pathogenicity (yellow). Flanking these are the viral inverted terminal repeats (ITR), which include the viral origin of replication and packaging signal. The latter sequences are cis-acting as they affect only genes on the same nucleic acid strand. B, The packaging construct, illustrated here as a plasmid, includes the gene required for the production of viral structural proteins (red) and viral replication (blue). These genes are trans-acting as they need not be located on the same nucleic acid strand as the genes that they regulate. C, The recombinant vector contains a transgene cassette with its own transcriptional regulatory elements. This is reproduced and then packaged in the presence of the appropriate viral genes (supplied in trans from the packaging construct) as it is flanked by the necessary cis-acting regulatory sequences. The genes involved in viral pathogenesis are eliminated.

has a unique set of properties that lend themselves to specific applications. For example, RV and LV vectors undergo genomic integration and are therefore favored for applications requiring long-term expression, whereas AdV vectors do not undergo integration and remain as episomes that are rapidly lost in replicating target cell populations with resultant transient expression.

2. Strategies in transplantation gene therapy Efforts thus far at gene therapy in solid organ transplantation have been confined to animal models. In broad terms,

2 strategies have been pursued: modifying the graft and modifying the host or recipient. 2.1. Graft modification Various gene delivery strategies, including both viral and nonviral tools, have been used for both ex vivo and in vivo graft manipulation. Although the graft or the recipient may be the focus of gene delivery, in many cases (particularly where the graft is delivering a factor systemically), gene delivery to the graft exerts its effect most potently through host modification. The chief goal of gene delivery to the graft is the amelioration of insults that are suffered by a transplant

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as a result of adaptive immune responses, and those that result from innate immune damage, particularly when associated with ischemia and reperfusion injury. 2.1.1. Modulation of co-stimulatory pathways Blockade of co-stimulatory pathways has been extensively explored in many models. The CD28/B7 costimulatory pathway has been targeted using a construct consisting of cytotoxic T-lymphocyte associated antigen 4 (CTLA4) combined with immunoglobulin G. The resulting protein (CTLA4-Ig) is soluble and has a prolonged half-life compared to CTLA4. Using mostly AdV vectors and CTLA4-Ig, prolonged allograft survival has been achieved in rat liver [24,25], rat heart [26-28], rat kidney [29], rat cornea [30], mouse and rat pancreatic islet [31,32], rat lung [33], and rat hind limb models [34]. CTLA4-Ig gene therapy has also been successful in achieving prolonged graft survival in a number of concordant xeno-transplant models including rat-to-mouse islet grafts [35], rat fetal cardiomocytes transplanted into mice [36], and hamster-to-rat liver transplants [37]. In a number of studies, CTLA4-Ig gene therapy has been shown to have synergistic effects with other agents such as immunosuppressive drugs [38], donor leukocyte infusion [39], or concomitant CD40-Ig gene therapy [34,40,41]. The CD40/CD154 co-stimulatory pathway has been a target of gene therapy via the expression of a CD40-Ig fusion protein. Delivered mostly by AdV, this has been successful in prolonging survival of allografts including those of rodent liver [42,43], rodent heart [40,44], hind limb [34], and pancreatic islets [45]. This approach has also been used

successfully in rodent xenograft models of heart and islet transplantation, when combined with immunosuppression (FK779) [46] or concomitant CTLA4-Ig gene therapy [41], respectively. When activation of these co-stimulatory signals has been blocked, the activity of other pathways such as the inducible co-stimulator–B7 related protein-1 pathway has been identified as important in transplant rejection [47]. Although gene therapy to block these additional stimulatory pathways has not so far been successful [48], another pathway that has been targeted with some success is the inhibitory interaction between Programmed Death 1 (PD1) and its ligand Programmed Death 1 Ligand 1 (PD-L1). Heart allografts modified to express a PD-L1-Ig fusion protein have a slight survival advantage over untransduced allografts. This effect was synergistic with low dose immunosuppression [49]. 2.1.2. Manipulation of cytokine expression Certain cytokines or their receptors, particularly interleukin (IL)-10, a virally derived analogue of IL-10 (vIL-10), transforming growth factor β (TGF-β), the p40 subunit of IL-12, or soluble IL-1 receptor, have all been delivered to a graft in the context of transplantation. Interleukin 10 has been the most extensively examined, and there have been relatively smaller numbers of studies focusing on the use of other cytokines for organ modification (Table 2). IL-10 or vIL-10 have produced mostly modest improvements in graft survival in models including nonvascularized cardiac allografts [53,54,63,64], vascularized heterotopic cardiac allografts [52,65-71], liver allografts [72-74], corneal allografts [75,] and pancreatic allografts [76].

Table 2 Studies examining the effects of cytokines for organ modification Gene

Modality of gene therapy

Outcome

Model

Reference

TGF-β

Liposomal transfection and AdV AdV

Mouse vascularized cardiac allograft

[50]

Rat liver allograft

[51]

Rabbit heart allograft Mouse nonvascularized cardiac allograft Mouse vascularized cardiac allograft Human islets transplanted into NOD/SCID mice

[52] [53,54] [55] [56]

Rat in situ I/R model

[57]

Rat vascularized cardiac allograft

[58] [59]

[62]

IL-1R type 2 Ig fusion protein

AdV

IL-13

AdV

P40 subunit of IL-12

RV

Prolonged allograft survival only when combined with CD8+ depletion Reduced TNF-α and IFN-γ production Prolonged allograft survival Prolonged allograft survival Prolonged allograft survival Improved graft function when combined with vascular endothelial growth factor Improved survival and function after ischemia/reperfusion injury Improved survival when combined with immunosuppression Improved survival and function after ischemia/reperfusion injury Prolonged survival

IL-18 binding protein Ig fusion protein IL-17 receptor Ig fusion protein

AdV

Prolonged allograft survival

Rat in situ and syngeneic transplant I/R model Mouse myoblast cell line transplanted into allogeneic strain Rat vascularized cardiac allograft

AdV

Prolonged allograft survival

Rat vascularized cardiac allograft

IL-2 IL-1 receptor antagonist

AdV Plasmid transfection ASO knockdown AdV

AdV or liposomal

TNF-α indicates tumor necrosis factor α; IFN-γ, interferon γ; ASO, antisense oligonucleotide; I/R, ischemia and reperfusion.

[60] [61]

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Table 3 Studies examining the effects of expression of leukocyte adhesion molecules and chemokines Gene

Modality of gene therapy

Outcome

Model

Reference

vMIP-II and MC148

Plasmid transfection

Prolonged allograft survival

[93]

ICAM-1

Hyperbaric injection of ASO leading to ICAM-1 knockdown ASO injection leading to ICAM-1 knockdown Hyperbaric injection of ASO leading to ICAM-1 knockdown LV expressing chemokine antagonist RANTES 9-68 AdV expressing NH(2)-terminal deletion mutants of MCP-1 and RANTES

Reduced ischemia and reperfusion injury Prolonged survival

Mouse vascularized and nonvascularized cardiac allograft Rat heterotopic cardiac allograft Mouse islet allograft

[90]

RANTES MCP-1 and RANTES

[89]

Prolonged survival and reduced chronic rejection Prolonged survival

Rat heterotopic cardiac allograft

[94]

Rat heterotopic cardiac allograft

[91]

Prolonged survival

Rat heterotopic cardiac allograft

[92]

ASO indicates anti-sense oligonucleotide; MCP-1, monocyte chemoattractant protein 1.

In the context of lung allografts, improved function after transplantation was observed using IL-10–mediated gene therapy [77-81]. Although IL-10 gene transfer to pancreatic islet transplants produced prolonged survival when returned to NOD syngeneic hosts [82,83], no protection was afforded when transplanted to allogeneic recipients [84]. The modest allograft survival prolongation achieved by IL-10 gene therapy when used alone was greatly augmented by the addition of concomitant IL-4 gene therapy [85]. A variety of mechanisms may contribute to the graft protection afforded by the local expression of IL-10 or its viral analogue. Graft IL-10 or vIL-10 expression reduces infiltration by both CD4+ and CD8+ T cells [54,65,67], while decreasing the expression of proinflammatory cytokines, such as interferon γ and tumor necrosis factor [67,77]. Interleukin 10 increased the apoptosis of alloreactive CD8+ T cells infiltrating the graft possibly through activationinduced death via the Fas/Fas-L and Bax pathways [86,87]. The protection of aortic allografts from chronic rejection afforded by IL-10 gene therapy was shown to be mediated by reduced neointimal proliferation mediated through IL-10– dependent up-regulation of hemoxygenase 1 (HO-1) [70]. Interleukin 10 is known to induce Treg [88], and it is tempting to speculate that the effects of gene therapy using IL-10 may involve this mechanism. However, there is no direct evidence to support such a contention. 2.1.3. Leukocyte recruitment and adhesion A number of studies have focused on modulating rejection by altering leukocyte tethering to the inflamed endothelium of the transplanted tissue. In particular, attempts have been made to disrupt the interaction between intercellular adhesion molecule 1 (ICAM-1) and its integrin receptor, lymphocyte function-associated antigen 1, using antisense oligonucleotide knockdown of ICAM-1 expression. This has been shown to reduce ischemia and reperfusion injury associated with heterotopic rat heart allografts [89], prolong survival of mouse islet allografts [90], and reduce chronic rejection of heterotopic rat heart transplants. Attempts have been made to disrupt the

ability of chemokines to attract and activate leukocytes to the site of inflammation within the graft by using antagonists of chemokine-mediated attraction. Specifically, antagonists of the CC-chemokine regulated on activation, normal T cell expressed and secreted (RANTES) [91,92], and viral chemokine antagonists viral macrophage inflammatory protein II (vMIP-II) and MC148 [93], have been delivered using gene therapy techniques to prolong survival of heterotopic heart allografts (Table 3). 2.1.4. Immunomodulatory and cytoprotective enzymes Attempts have been made to use gene therapy to exploit the immunomodulatory and cytoprotective capacity of enzymes including indoleamine dioxygenase (IDO), HO-1, nitric oxide synthetase, superoxide dismutase, and catalase. Indoleamine dioxygenase expression has been shown to slightly prolong the survival of corneal allografts [95] and to attenuate acute injury [96] and chronic fibrosis [97] after lung allo-transplantation. However, high-level expression of IDO in liver using AAV has no effect on liver transplant rejection [98]. Nitric oxide synthetase gene transfer to arterial allografts protects them from rejection [99-101]. Hemoxygenase 1, when administered systemically via an AdV vector, produces long-term survival of rat heart allografts [102]. Adenovirus HO-1 gene transfer protects against I/R injury in steatotic rat livers by preventing apoptosis of injured cells and prolongs rat liver allograft survival modestly [103-107]. Hemoxygenase 1 gene transfer using an AdV vector protects the function and viability of human islets in culture [108] and ameliorates chronic rejection of rat aortic allograft [109]. Superoxide dismutase delivered via an AdV vector attenuates I/R injury and, in rat liver transplantation models, improves survival of recipients of normal and fatty livers [110,111]. Adenovirus transfer of the catalase gene reduced susceptibility to oxidative stress in vitro [112]. After delivery by polylipid nanoparticles, the genes for both superoxide dismutase and catalase were shown to ameliorate the effects of warm in situ ischemic injury in the mouse liver [113].

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2.1.5. Apoptosis pathways A number of studies have examined the delivery of genes to induce apoptosis of infiltrating alloimmune cells. The expression of Fas-L has produced results that are disparate and difficult to reconcile. Modest prolongation of rat kidney allograft survival was achieved after graft transduction by an AdV expressing Fas-L [114]. Syngeneic myoblasts expressing Fas-L co-transplanted with islet allografts were reported to prolong islet allograft survival in a site-specific manner [115]. However, when pancreatic islets themselves expressed Fas-L (either after AdV transduction or in a transgenic model), those cells were rapidly destroyed by a lymphocyte-independent immune response dominated by IL8 expression and the recruitment of granulocytes [116,117]. This was not a phenomenon affecting pancreatic islets exclusively, as heart grafts expressing Fas-L, even when transplanted into syngeneic recipients, were subject to a rapid and destructive inflammatory response [118]. These apparently contradictory results may be partly explained by differing levels of Fas expression on the parenchymal cells of different organs (parenchymal cells expressing Fas being susceptible to fratricide or suicide upon Fas-L co-expression) and partly by the narrow therapeutic window for this gene [114,119]. These results may also reflect engagement of pattern-recognition receptors in some, but not all, cell types as an unintended consequence of viral transduction. Fas–Fas-L interactions have also been targeted indirectly by gene therapy with IL-10, which promotes apoptosis of alloreactive T cells by increasing their expression of Fas [69,87]. Bcl-2, Bcl-xl, and A20 are antiapoptotic genes that may protect cells targeted by an alloimmune response from nuclear factor κB–mediated immune activation and apoptosis [120,121]. There is evidence to suggest that the combination of A1 with A20 may have a synergistic protective effect [122]. AdV transduction of the liver with Bcl-2 improved its resistance to damage with prolonged cold storage and ex vivo rewarming [123], and transfer of Bcl-xl to the corneal endothelium by a lentiviral vector has been shown to prolong allograft survival [124]. Gene therapy with A20 aids the resistance of pancreatic islets to the insult of procurement and transplantation and results in cure of diabetes in syngeneic diabetic mice with a smaller inoculum of islet cells than usually required [125]. 2.2. Host modification Host modification strategies aim to render a recipient more amenable to accepting an allograft. Many of these techniques are based on exposing the recipient to donor major histocompatibility complex (MHC) encoded by a vector aiming to mimic the effects of donor-leukocyte infusion. Conversely, some groups have sought to enhance the tolerance-inducing capacity of donor cells by genetic modification of donor antigen-presenting cell (APC) (particularly dendritic cell [DC]). This strategy seeks to modify the host immune response without genetic modification of the graft.

2.2.1. Donor MHC transfer The goal of donor MHC transfer is to modify autologous cells to encode donor-type MHC proteins. This may allow the realization of the potential of tolerance to those proteins without the risk of graft-versus-host disease that is associated with tolerance induced by allogeneic bone marrow transplantation [126]. Retroviral transduction of autologous bone marrow to express a class I MHC antigen from an allogenic strain induced specific tolerance to skin grafts derived from animals of that strain by T-cell anergy rather than by clonal deletion [127-129]. Similarly, AdV transfer of class I MHC antigen resulted in long-term survival of heart allografts from the same strain when combined with transient CD4+ T-cell depletion [130]. Introduction of allogeneic MHC class I genes into the thymus either by direct vector injection [131] or by injection of transfected autologous cells [132] produced donor-specific tolerance to subsequent allografts. Transduction of a heart allograft recipient's native liver with an AdV vector expressing soluble allogeneic MHC class I antigen modestly prolonged the survival of subsequent heart allografts sharing the MHC class I antigen encoded by the AdV vector [133]. Delivery of a gene expressing allogeneic MHC class II antigens by retroviral vector into autologous bone marrow cells prolonged survival of allografts sharing the same antigen as the transduced bone marrow but differing at other MHC loci [134,135]. One of the major limiting factors with MHC gene therapy is the maintenance of sufficient autologous cells expressing the allogeneic MHC to achieve durable tolerance. This has been addressed by the addition of a selection marker to the vector expressing allogeneic MHC, enabling selective survival of transduced stem cells and ensuring persistence of allograft tolerance [136]. 2.2.2. Transfer of transduced donor antigen-presenting cells APC can act to augment or down-regulate an immune response depending on the available second signals and the prevailing state of immune activation [137]. The process of solid organ transplantation is intrinsically immunogenic, being associated with ischemia-reperfusion injury and other insults to the graft and recipient. This tends to skew the profile of DC away from a tolerogenic program in a number of ways, including an increase in the expression of costimulatory molecules interacting with the T cell [138]. Viral transduction per se (particularly when AdV vectors are used) can also render APC more immunogenic. Modification of the APC using gene therapy aims to turn the APC toward a tolerogenic program, counteracting the immune activating effects not only of transplantation but also of viral transduction on these cells. A number of studies have modified DC and demonstrated a capacity for reduced allostimulation in vitro from DC expressing a range of transgenes including CTLA-4 [139], vIL-10 [140], IDO [141], and Fas-L [142]. The expression of TGF-β was able to counteract the immunostimulatory effect of AdV transduction on DC [143]. Using donor-derived DC modified

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to express IL-10 [144], Fas-L [145], or CTLA-4 [146], the survival of small bowel, heart allografts, and islet allografts, respectively, was modestly prolonged. Conversely, the expression of IL-4 by allogeneic DC augmented immune responses in vitro and accelerated allograft rejection [147]. 2.2.3. Transfer of transduced donor lymphocytes T lymphocyte and natural killer cell subpopulations modulate immune homeostasis and have also been shown to contribute to tolerance induction in transplantation [148]. Markers, such as CTLA4, GITR, and FoxP3, have enabled the identification of T-regulatory cells, and gene therapy has been applied to their generation for tolerance induction in preclinical transplantation studies. Chai et al [149] used retroviral vectors encoding FoxP3, a transcription repressor protein important for regulatory cell function, to transduce T lymphocytes transgenic for the male specific H-Y antigen. Adoptive transfer of the resultant population to female mice significantly prolonged the survival of male skin grafts. In an alternative approach, Tsang et al [150] transduced regulatory T lymphocytes with a retrovirus encoding a TCR specific for an allogeneic peptide presented by MHC class II molecules of the recipient APC. This conferred the regulatory cells with specificity to transplanted cardiac grafts which, in combination with rapamycin and anti-CD8 antibody treatment, significantly enhanced the survival of fully mismatched cardiac grafts. Although highly artificial in their design, these studies demonstrate proof-of-principle for exploitation of gene therapy and regulatory T cells to improve transplantation outcomes in the clinic.

3. Potential clinical application of gene therapy in transplantation Over more than a decade, experimental animal studies have clearly demonstrated success in overcoming both immune and non-immune barriers to organ transplantation using a wide variety of candidate genes, delivered by a range of vectors, using a number of different strategies. However, there are many obstacles to be overcome before these advances can be translated into a treatment that would improve outcomes in human clinical transplantation. Gene therapy has found most ready application in human diseases where the outcomes are currently very poor and conventional treatments either highly toxic or relatively ineffective, or both. Examples included advanced malignancy or severe genetic disorders [151]. However, early human gene therapy trials have suffered a number of well-publicized adverse events. During the trial of an AdV vector to treat a partial ornithine transcarbamylase deficiency, an 18-year-old study participant died of a severe systemic inflammatory response to components of the vector. The severity of this response had not been anticipated based on preceding animal studies or even previous experience in the same study with lower

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doses of the vector [152]. Another major setback in human gene therapy clinical trials was the development of malignancy in an infant successfully treated for X-linked severe combined immunodeficiency using retroviral gene therapy [153] due to an insertional mutagenesis event. This complication was believed before the trial to be a remote possibility, but malignancy was eventually reported in 4 of 9 patients treated in this trial [154] and in 1 of 10 in a second similar trial [155]. It is therefore likely that only once gene therapy has been successfully established as a safe and effective treatment in another area of medicine that it can be advanced as an alternative or adjuvant to current transplantation protocols. The clinical scenario in which gene therapy is likely to be most appropriate is that of deceased organ donation. Here, it may be feasible to deliver a recombinant vector to a whole organ before implantation either by ex vivo manipulation or by delivery in vivo before organ procurement from the donor. Nevertheless, there are a number of issues surrounding the clinical applicability of these techniques. Given that the different organs have varying tropisms for different vectors and promoter systems [22], it is unlikely that a single vector would be ideal for all the commonly transplanted tissues. Ex vivo gene delivery is a way of avoiding exposure of other organs to a vector or segregating organs such that the most ideal vector system can be used for each tissue. However, perfusion of the cold preserved organ is likely to be less effective than in vivo gene delivery [11] and thus not the preferred technique from the point of view of transduction efficiency. Another issue is the relatively slow kinetics of gene expression mediated by recombinant vectors. For example, although AAV is an otherwise promising vector, maximal expression is achieved only some weeks after vector administration in animal models [22]. The average length of stay in an intensive care unit for a deceased donor is around 3 days [156], and the time from the diagnosis of brain death to organ procurement is even less. Thus, the kinetics of AAV vectors are incompatible with this clinical scenario. However, variant AAV vectors are now available that may help circumvent the requirement for complementary DNA strand synthesis and provide not only more rapid onset of transgene expression [157] but also more efficient transduction [158]. Such self-complementary vectors may enhance the prospects for the clinical applicability of AAV in the context of transplantation. Although AdV vectors achieve more rapid transgene expression than AAV vectors [22], AdV administration causes activation of host parenchymal and myeloid cells independent of AdV gene transcription. The myeloid cells in particular release chemokines, both locally and systemically, in response to AdV transduction [159,160]. This response is associated with cellular inflammation and even fatal systemic inflammatory responses [58]. Not only does this feature of AdV pose safety risks, but it limits the long-term expression of the AdV-encoded genes.

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4. Conclusions Any novel treatment must offer real advantages with low risk of adverse events. As described above, the possibility of unanticipated complications has been realized with previous attempts to use gene therapy to treat genetic disease. Although many animal studies have been performed using gene therapy in transplantation, most have shown only a modest effect. In some studies, gene therapy has been augmented by the addition of subtherapeutic immunosuppressive drug treatment [38,46,49,58]. However, this approach may also increase the risk of complications (such as malignancy or infection) associated with the use of viral vectors. The latter possibility may hamper the prospects for a graded introduction of gene therapy technologies combined with current treatments. Therefore, the greatest barrier to advancing gene therapy into the clinic is the very success of the current practice of transplantation. This work was supported by the National Health and Medical Research Council of Australia and the Microsearch Foundation of Australia. JML was supported by a scholarship from the Northcote Foundation. No authors have any conflict of interest to declare.

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