Strategies for Local Gene Therapy of Corneal Allograft Rejection

Abstract

Penetrating keratoplasty is the most common type of tissue transplant in humans. Irreversible immune rejection leads to loss of vision and graft failure. This complex immune response further predisposes future corneal transplants to rejection and failure. A diverse armamentarium of surgical and pharmacologic tools is available to improve graft survival. In this review, we will discuss the various gene therapeutic strategies aimed at potentiating the anterior chamber-associated immune deviation to extend graft survival.

INTRODUCTION

Penetrating keratoplasty (PKP), a highly successful tissue transplant procedure, is performed worldwide for many indications, e.g. visual rehabilitation, improvement of tectonic support, or removal of infectious agent(s). Tissue shortage and the current transition to lamellar keratoplasties to preserve healthy tissue have modulated the numerical success of PKP.^1–3 Its long-term success is limited by graft failures and allograft rejection accounts for up to 40% of all corneal transplant failures.^4–10 A wide variety of premorbid risk factors, such as ocular inflammatory diseases, corneal neovascularization, prior graft failures, and glaucoma, increase the predisposition to allograft failure.³,⁹,^11–14 A broad spectrum ^15–20 of strategies has been proposed to prolong graft survival with protean success: tissue matching, reduction of antigenic factors, immunosuppression, lamellar transplantation, penetrating limbo-keratoplasty, human amniotic membrane transplantation, tissue engineering, and gene therapy.

Gene therapy is a potent modality that promises remedies to many inherited and acquired diseases. The unique features of the cornea and the eye present a splendid platform for gene therapies, particularly modulation of allograft rejection. These properties include stable ex vivo storage of donor tissue, ex vivo and in vivo accessibility, transparency of the cornea, and immune privilege of the eye. This review, however, will focus only on gene therapy to prolong PKP graft survival. We will describe the immune privilege of the eye, strategies to maintain this immune privilege, and the relevant literature.

IMMUNOLOGY OF CORNEAL GRAFT REJECTION

PKP enjoys a higher success rate than other tissue and organ transplantations, despite the routine human leucocyte antigen (HLA-) matching. Indeed, the Collaborative Corneal Transplantation Studies Research Group concluded that neither HLA-A, -B, nor -DR antigen matching substantially reduces the likelihood of graft failure.¹⁵ This remarkable property is achieved by maintenance of the integrity of the ocular immune privilege. Notwithstanding, the long-term survival rate remains unsatisfactory, especially in high-risk recipients.¹,¹³ Irreversible allograft rejection precipitates 16-30% of all corneal graft failures.⁵,¹⁹ The prognosis worsens substantially with subsequent grafts, and the survival rates for third and fourth regrafts diminish to 25% and 0%, respectively.⁸,¹² Allograft rejection is an labyrinthine, multifactorial process that requires the recognition of a foreign tissue by the host, initiation of the immune response cascade, and subsequent destruction of the foreign tissue. The literature substantiates a cell-mediated immune response as the primary mechanism involved in corneal allograft rejection; however, current evidence also suggests that alloantibodies play a role as well. Both direct and indirect antigen presentation appear to be involved in the sensitization phase, followed by activation against both major and minor histocompatibility antigens that are resident on the corneal graft. The three-legged stool analogy of the immune privilege is depicted in Figure 1. Figure 1 also outlines potential targets for gene therapy.

Figure 1.

Gene therapeutic strategy for corneal allograft rejection

MAF: Macrophage activation factor; MIF: migration inhibition factor, APCs: Antigen-presenting cells, NK: Natural killer cells, vMIP II: viral macrophage inflammatory protein, NGF: Neuronal growth factor, PDGF: Platelet-derived growth factor, FGF: Fibroblast growth factor, INF: Interferon, TNF: tumor necrosis factor, SDF: Stromal-derived factor, PEDF: Pigment epithelium-derived factor, msTie2: murine-soluble Tie2, msFLK1: murine-soluble vascular endothelial growth factor receptor-2, IDO: Indoleamine 2,3 dioxygenase, IL: Interleukin, TGF: Transforming growth factor, TNFR: Tumor necrosis factor receptor, DAF: Decay accelerating factor, ICOS: Inducible Co-stimulator, Stat 3: Signal transducers and activators of transcription

Note: Green arrows and (+) signs denote positive interaction / upregulation. Red arrows and (-) denote negative interaction / downregulation.

Afferent arm

Similar to the human brain and testes, the cornea is an immunologically privileged site that is protected by multiple mechanisms. The avascular cornea maintains its immune privilege by the reduced expression of the major histocompatibility complex (MHC) class I and absent expression of MHC class II on antigen-presenting cells (APCs), by the lack of Langerhans cells in the epithelium, and by the expression of Fas ligand (FasL) by endothelial cells. FasL triggers apoptosis of Fas+ neutrophils and T cells that infiltrate the anterior chamber. Importantly, the anterior chamber allows the corneal allograft to induce deviation of systemic delayed-type hypersensitivity. This afferent blockage is violated by inflammation and neovascularization. Inflammatory stressors induce MHC II expression by keratocytes, bone marrow-derived stromal cells, and endothelium, thereby assisting antigen recognition by the host. The inflammatory cytokines also induce hemangiogenesis and lymphangiogenesis, further potentiating the rejection cascade.

Vascularization expedites the penetration of class II MHC+ Langerhans cells and centrifugal migration of allograft passenger leukocytes into the host tissue, the subsequent transformation into allostimulatory APCs, and the interaction with T-cells in the conjunctival associated lymphoid tissue and/or regional lymph nodes. This cascade vitiates the integrity of the anterior chamber immune privilege, thereby worsening the prognosis of subsequent transplant. Clinically, corneal neovascularization is a significant risk factor for graft rejection.³,⁹,^11–14 One study¹² reported that endothelial allograft reactions increased from 3.8% to 36.7% in patients with preoperative corneal vascularization; and 90% of allograft rejection reactions occurred within 1 year after transplant.

Consequently, neovascularization has generated profound interest, not only for corneal graft rejection, but also for tumorigenesis or organogenesis. Neovascularization has been associated with upregulation of growth factors. Angiogenesis suppression demonstrates its powerful utility in the arsenal against many ocular diseases, such as neovascular age-related macular degeneration, proliferative diabetic retinopathy, neovascular glaucoma, or retinopathy of prematurity. In addition to HLA-typing and inhibition of the inflammation cascade, pre-sensitization to activate the anterior chamber-associated immune deviation and anti-vascularization strategies such as anti-vascular endothelium growth factor receptor (anti-VEGFR), photodynamic therapy, and needle diathermy have all been proposed to improve allograft survival. We will discuss important advances in gene therapy for afferent blockage in the following sections.

Central processing stage

The central stage includes activation, differentiation, and proliferation of lymphocytes. The CD4+ T cells appear to be the principal conductors of the cell-mediated immune response. T helper 1 (Th1) cells orchestrate mononuclear cell infiltration and production of interferon (INF)-γ and tumor necrosis factor (TNF)-α, whereas Th2 cells are involved in the production of interleukin-4 (IL-4), IL-5, and IL-13 and transplant tolerance. Th17 cells are thought to be induced by transforming growth factor (TGF)-β, IL-6, and IL-21 via the orphan nuclear receptors and the retinoid-related orphan receptor gamma tau. These cells disseminate cytokines such as IL-17, IL-17F, IL-21, and IL-22. Interleukin-17 is a proinflammatory cytokine that involves chemokine expression, leukocyte infiltration, and tissue inflammation. Its participation in allograft rejection in cardiac, lung, renal, and vascular transplantations has been documented. Accordingly, therapeutic targets include T helper (Th) cells and cytokine production cascades as well.

Inhibition of the central processing stage can be accomplished mainly by using monoclonal antibodies directed against antigen presentation or T-cell activation steps, anti-interleukin 2, calcineurin inhibitors, and antimetabolites. Immunosuppressive targets for gene therapy currently being explored include CD4, CD8, T-cell receptors, and costimulatory molecules such as CD80, CD86, CD154, and CD25. Nonetheless, these steps typically occur centrally, and local transgene products may be inadequate. A recent study designed to investigate the effects of cervical and thoracic lymphadenectomy on corneal graft rejection in inbred rats failed to demonstrate any difference.²¹ The authors suggest that sensitization and clonal expansion of corneal alloantigen-reactive cells may not be limited only to the regional lymph nodes (superficial cervical, facial, internal jugular, and posterior cervical) in rat. Thus, therapies may need to target both locally and systemically.

Efferent arm

The efferent arm is, effectively, the destruction of the allograft by the cellular and humoral effector mechanisms. The cornea exercises efferent blockage by multiple mechanisms. Graft membrane-bound and host anterior chamber-soluble molecules can neutralize immune effector elements at the host-graft junction and the anterior chamber. Two notable factors are the decay-accelerating factor (DAF, CD55) and Fas ligand (FasL, CD95). DAF expression has been detected in lacrimal gland acinar cells, ocular surface epithelium, corneal endothelium, trabecular meshwork, and retinal pigment epithelium. It is a membrane complement regulatory protein that protects cells from autologous complement activation, formation of the membrane attack complex, and cell lysis.²²,²³ Similarly, FasL preferentially induces apoptosis of neutrophils and effector T cells; corneal epithelium and endothelium demonstrate constitutive expression of Fas ligand (CD 95) as a self-defense mechanism against neutrophils and activated T cells.

During an acute rejection, elevated concentrations of many cytokines are detected in lacrimal secretion, the anterior chamber, and the cornea. One study²⁴ reported a high concentration of IL-6 and IL-8 in the tears of patients with concomitant corneal graft rejection, whereas expression of IL-10, tumor necrosis factor (TNF)-α, and IL-12p70 abated. Elevation of IL-6 was observed in aqueous humor and IL-8 was detected in corneal tissue.²⁵,²⁶ Similarly in a rat model, increased levels of IL-1β, IL-6, IL-10, IL-12 p40, and the macrophage inflammatory protein (MIP)-II were documented in both syngeneic and allogeneic recipients during the initial phase of rejection. The cytokine levels regressed to the pretransplant levels by week 2 after the onset of allograft rejection in the syngeneic group, but remained elevated in the allogeneic group. Of note, T cell-derived cytokines IL-4, IL-13, and interferon (INF)-γ were detected only during the rejection phase in allogeneic recipients. Migration of Langerhans cells expressing class II MHC into the cornea is mediated by IL-1 and TNF-β. TNF-α is responsible for proinflammatory and immunoregulatory functions; it upregulates adhesion and costimulatory molecules and activates the NK-kB signal pathway. Interleukine-10 is a potent immunomodulatory cytokine that interacts with APCs and inhibits production of monokines such as IL-1, IL-6, IL-8 and TNF-α. Recent discoveries in gene therapy for efferent blockage are discussed later.

GENE THERAPY

Irreversible immunological rejection is the leading cause of corneal transplant failure. Multidisciplinary pharmacologic strategies have been advocated to reduce failure. Topical or systemic corticosteroids typically constitute the first line of defense. Corticosteroids provide broad-spectrum immunosuppression by suppression of APCs, T lymphocytes and other cells, as well as inhibition of cytokine production. Systemic immunosuppression regimens are more commonly reserved for high-risk grafts. Systemic calcineurin inhibitors, antimetabolites, or monoclonal antibodies have been employed, either as monotherapy or in combination with corticosteroids, with variable success. Systemic cyclosporine has shown inconsistent results in prolongation of survival in high-risk patients and, in fact, may induce severe adverse effects, such as systemic hypertension, renal deficiency, and malignancy. Use of a multi-agent regimen has been proposed to leverage the synergistic mechanisms to achieve maximal therapeutic effects and minimal adverse effects in these high-risk transplants.¹⁷

Gene delivery methods

Corneal grafts constitute an excellent platform for gene therapies. Donor corneas are readily available for ex vivo manipulation. Both corneal endothelium and epithelium are accessible for broad range of in vivo and in vitro transduction techniques. Ex vivo gene transfer presents tantalizing opportunities, as the excess vectors and culture media may be eliminated to minimize the risks of vector spread and xenobiotic reaction; quality control measures can be implemented. A diverse selection of in vivo modalities encompasses topical instillation, intracorneal injection, ballistic delivery, electroporation, ultrasound-assisted, etc. Transgenes can be administered with naked plasmid, viral vector, or nonviral vectors. Naked plasmid offers the advantages of less associated inflammation and less concern for xenobiotic reaction; but the disadvantages include short-term stability and gene expression and poor penetration through intact epithelium, and typically, they must be delivered by direct intracorneal injection. Viral vectors include adenovirus, adeno-associated virus, herpes virus, retrovirus, lentivirus, baculovirus, etc. And nonviral vectors encompass gold particles, liposomes, chitosan, biocompatible and degradable polymers, and dendrimer.

Ensuring effective local concentration is a fundamental challenge in pharmacology. Ocular gene delivery is facilitated by the ease of access; and instantaneous administration can be achieved by direct topical instillation, subconjunctival injection, and intracameral or intravitreal injection. For topical application, precorneal factors such as small load, poor conjunctival, corneal, or scleral penetration, or mechanical elimination by tears and blinking can produce suboptimal bioavailability and reduce transfection efficiency. Clearance via the lacrimal drainage system may also lead to systemic absorption and toxicity, especially if a viral vector is employed. Inherent risks exist with intracameral or intravitreal deliveries such as endophthalmitis, retinitis, or retinal tear or detachment.

Other modalities are being developed to improve delivery and reduce adverse effects. A helium-driven ballistic gene gun, used to deliver plasmid-coated gold microparticles, has been available for more than 10 years. Gene delivery efficacy and the extend of tissue injury depend on the size of the microparticles and the cornea-gene gun interface.²⁷ Use of a pegylated liposome showed promising data for green fluorescent protein (GFP) cDNA delivery in rabbit and rat models.²⁸,²⁹ Liposome can also be utilized for endothelium regeneration; Luo et al.³⁵ performed lipofectine-mediated delivery of plasmid pcDNA4 for platelet-derived growth factor (PDGF)-B in cultured cat corneal endothelial cells and detected increased expression of PDGF. Femtosecond laser-assisted delivery of HIV1-derived lentiviral vector has been reported to result in more efficient transduction of the porcine stromal keratocytes compared to direct intracorneal injection.³⁰ Given the inflammatory response incited by femtosecond laser, it will be interesting to see whether this technique will actually influence long-term allograft survival. Chitosan-modified adenoviral vector has been used in primary cultures of bovine and rat corneal epithelial cells.³¹ More recently, Klausner et al.³² reported the successful transduction of rat keratocytes after intracorneal injection of chitosan nanoparticles to deliver a DNA sequence encoding a marker protein. Delivery of plasmids encoding eGFP or β-gal markers by electroporation showed good transfection efficiency, with low early endothelial cell death in preserved human cornea.³³

Gene therapies

It has been reported that gene transduction and expression might be different in in vivo or ex vivo compared to in vitro experiments. Accordingly, although some in vitro tissue culture studies will also be discussed throughout this review and summarized in Tables 1 and 2, the bulk of the discussion will be focused on animal orthotopic keratoplasty models, as tabulated in Table 3.

Table 1.

Gene delivery using cultured tissues

Table 2.

Gene delivery using ex vivo and in vivo models

Table 3.

Gene delivery using orthotopic allograft animal models

Angiogenesis suppression

Suppression of angiogenesis is a well-documented strategy. Angiogenesis inhibitors that have been identified and explored for medical applications include prolactin, endostatin, angiostatin, somatostatin, cyclosporin A, the fumagillin analog TNP-470, steroids, nonsteroidal anti-inflammatory agents, thalidomide, methotrexate, and even cultured supernatant of human amniotic cells. Important advances in gene therapy have enabled us to evaluate angiosuppressive modalities for graft failure using animal models.

Diverse delivery methods have been tried to introduce an anti-angiogenic gene for corneal diseases. Direct instillation of anti-mouse SDF-1α antibody was found to decrease intracorneal VEGF and C-Kit expression and the resultant alkali-induced corneal neovascularization in a mouse model.³⁴ In a RCS-rdy + rat model, direct injection of adenovirus carrying VEGF cDNA into the anterior chamber reduced corneal neovascularization induced by silver nitrate or potassium nitrate cauterization.⁴⁶ Naked plasmid delivery of Flt-1, the vascular endothelial growth factor (VEGF) receptor 1, to the corneal stroma has been shown to decrease VEGF-induced corneal neovascularization in the CD-1 mouse.⁵² Subconjunctival injection of siRNAs targeting the VEGF pathway in BALB/c mice decreased VEGF production and ocular angiogenesis induced by CpG and herpes simplex virus (HSV) infection.⁶¹ More recently, in a mouse corneal alkali burn model, subconjunctival injection of retroviral vectors for delivery of murine endostatin (mEndo), murine-soluble vascular endothelial growth factor receptor-2 (msFlk-1), or murine-soluble Tie2 (msTie2) caused overexpression of these antiangiogenic proteins and decreased corneal neovascularization.⁴⁹ Various non-viral vectors have been described as well.

Modeled after biological organelles, liposomal systems designed to encapsulate therapeutic agents for drug delivery have been found to have diverse ocular and systemic applications. In vivo delivery of BAI1-ECR, the extracellular region of brain-specific angiogenesis inhibitor gene, using liposome resulted in decreased corneal neovascularization in albino rabbits after epithelial denudement by n-heptanol and mechanical debridement.⁶² Yoon et al.⁶⁵ investigated subconjunctival delivery of GABPα/β plasmid using a liposomal vehicle in C57BL/6 mice. The authors discovered an initial decrease in in vivo VEGF and Robo4 expression with delayed neovascularization; nonetheless, after 2 weeks they found no significant difference in the mean neovascular area and number of vessels in the eyes treated with GABP versus those treated with empty vectors. Another group used lipofectine-mediated delivery of human pcDNA4-vascular endothelial growth inhibitor (pcDNA4-VEGI). In this silk-suture-induced corneal neovascularization rabbit model, Wang et al.⁶⁶ observed VEGI expression in the epithelium, stroma, and endothelium. More recently, plasmids encoding for endostatin mixed with non-liposomal lipid for subconjunctival injection were shown to attenuate VEGF expression and neovascularization in a rabbit alkaline burn model.⁶⁷

Other encapsulating materials include albumin, collagen, chitosan, and biodegradable polymers. Commercially available albumin, a major protein constituent found in blood and secreted by the kidneys, can be synthesized into nanoparticles for sustained gene delivery. Jani et al.⁶³ reported good results with intrastromal delivery of Flt-1, using albumin nanoparticles in a mechanical alkali trauma animal model. These authors found that the nontoxic albumin nanoparticles penetrated the corneal keratocyte cytoplasm; their presence was detected for at least 4 weeks and was correlated with reduced corneal neovascularization. Although synthesis of collagen nanoparticles has been described,⁸⁹ Bellner et al.⁶⁸ formed mixture of collagen and silencing hairpin RNA against heme oxygenase-2 (HO-2) instead. Subsequent to topical and subconjunctival deliveries, the authors noted delayed corneal wound healing, and increased angiogenesis and inflammatory cellular infiltration, identifying another target for anti-angiogenesis. Nanoparticles comprising of chitosan⁶⁹ or biocompatible and degradable polymer poly(d, l-lactic-co-glycolic) acid⁹⁰ have been reported as carriers for marker genes and antiangiogenic plasmid DNA pFlt23k.

Immunomodulatory gene therapy using orthotopic keratoplasty models

Recruitment of inflammatory cells is an intricate, multistep, coordination between cells of lymphocytic, granulocytic, and monocytic lineages, soluble mediators, and target cells. Suppression of the allogeneic rejection cascades often requires intervention at many levels. Multiple targets have been investigated and are discussed below.

IL-10 is an immunomodulatory cytokine that interacts with APCs and inhibits production of proinflammatory cytokines and TNF-α. Using a sheep model, Klebe et al.⁷² found prolonged survival of cornea transfected with cDNA encoding IL-10 after allogeneic transplantation. Another study⁶⁴ applied electroporation technique to drive gene expression after intracorneal injection of IL-10 plasmid in mice and found IL-10 expression with minimal evidence of inflammation.

Viral vectors have been researched extensively for gene delivery using cornea models. Gong et al.⁸⁰ investigating the role of viral IL-10 gene transfer using adenovirus and liposome, determined that neither mode of local ex vivo gene transfer led to prolonged allograft survival in a MHC class I/II mismatched rat model. Interestingly, the authors reported enhanced corneal allograft survival in host animals receiving systemic adenovirus IL-10 gene transfer and found increased mRNA expression in the draining lymph nodes but not in the corneal allografts, suggesting that IL-10 gene therapy modulation may occur more centrally. A study by Parker et al.⁸² observed that adenovirus has higher IL10 transduction efficiency compared to lentivirus. However, gene expression driven by lentivirus-SV40-IL10 in the human cornea was faster than in the ovine cornea and allograft survival was prolonged in the lentivirus-SV40-IL10-treated sheep recipients.

Interleukin-1 is a potent proinflammatory cytokine, associated with corneal neovascularization, chemotaxis of inflammatory cells, up-regulation of adhesion/costimulatory factors, and mediation of the acute phase response. Accordingly, the role of IL-1 receptor antagonist (IL-1 RA) in immunomodulation was investigated. It was found that IL-1 RA not only reduced the levels of cytokines, but it also decreased corneal neovascularization and improved graft survival in animal models. In a mouse model, transfection with IL-1 RA by direct injection of naked plasmid vector pIRES-EGFP-IL-1 RA resulted in downregulated expression of IL-1β and VCAM-1, as well as decreased corneal neovascularization and conjunctivalization.⁵⁸ Topical application of IL-1-RA was found to decrease all cytokine and chemokine levels, reduce corneal damage and cell infiltration, and improve corneal transparency.⁹¹ One study in MHC-disparate corneal transplant mice showed decreased rejection rates in the treated group as well.⁹²

Adenovirus delivered a gene encoding the soluble TNF-receptor fusion protein to rabbit corneal endothelium.⁷¹ Here, the authors detected good transgene expression in ex vivo corneal endothelial cells. However, only marginal improvement of graft survival was observed after orthotopic transplantation, compared to controls. Klebe et al.⁷⁶ showed that adenovirus delivery of P40 IL-12 prolonged corneal graft survival in a sheep orthotopic transplant model, whereas IL-4 induced eosinophilia, inflammation, and allograft rejection. Smad 7 encodes a nuclear protein that appears to inhibit the fibrogenic responses of keratocytes to TGF-β. It interacts with TGF-β receptor type-1 (TGFBR1), leading to degradation of both the protein and TGFBR1. In a rabbit orthotopic corneal transplant model, ex vivo gene transfer of Smad7 to corneal endothelial cells inhibited cellular loss and accelerated wound healing after PKP.⁸¹

Viral macrophage inflammatory protein (vMIP) is a chemokine analogue encoded by Kaposi’s sarcoma herpes-associated virus (human herpesvirus-8) that demonstrates in vitro antagonism to the chemokine receptors that control cell migration. In a murine model of corneal allograft rejection, transferrin-mediated liposome transfection of vMIP II gene plasmid showed prolongation of graft survival.⁸⁶ This was associated with decreased leukocyte infiltration of the corneal stroma.

A recent study exploited the roles of programmed death-ligand 1 (PD-L1) to inactivate lymphocytes and induce tolerance to corneal allografts.⁸⁷ Here, Nosov and colleagues introduced rat PD-L1 using lentivirus vectors into rat transplant model and found that PD-L1 attenuates rejection by modulating the expression of INF-γ and IL-6. The reduced expression of these proinflammatory cytokines correlated with decreased severity of cellular infiltration, lower corneal opacity score, and improved graft survival.⁸⁷

TNF-α stimulated gene/protein 6 (TSG-6), a multifunctional anti-inflammatory protein, has been reported to suppress a wide range of proinflammatory cytokines, such as INF-γ, IL-1β, IL-2, IL-6, IL-12, and myeloperoxidase (MPO). In this study, Oh et al.⁸⁸ performed intravenous injection of both human mesenchymal stem/progenitor cells (hMSCs) and TSG-6 in mouse model of corneal allotransplantation. The authors reported that most of the hMSCs were trapped in the lungs and, subsequently, activated to produce TSG-6. Overexpression of TSG-6 modulated the proinflammatory cytokines, reducing early inflammation, and late rejection of the corneal allografts. Immunostaining suggested decreased number of cells expressing class II MHC in the allograft button. Intravenous injection of TSG-6 achieved similar results. Given that hMSCs had been reported to be safe in randomized, double-blind, placebo-controlled trials in patients after acute myocardial infarction⁹³,⁹⁴, we anticipate further data on the use and long-term safety profiles of intravenous hMSCs and TSG-6 for corneal transplantation.

Co-stimulatory pathway using orthotopic transplant models

Traditionally, the key drivers of allograft rejection are Th1 and Th2 cells. Th1 cells mediate allograft rejection by releasing inflammatory cytokines and upregulating monocellular infiltration. Th2 cells are involved in the induction of transplant tolerance and production of IL-4, IL-5, and IL-13. Generally, IL-4, IL-6, and IL-10 activate Th2 cells and B-lymphocytes, tilting the Th1/Th2 response, because these cells are mutually inhibitory via cytokine production. Researchers aim to influence the Th1/Th2 balance and block the costimulatory pathway to prolong allograft survival.

A previous study⁹⁵ showed that anti-CD4 monoclonal antibodies delayed graft rejection in a rat model, whereas anti-CD8 monoclonal antibodies did not improve graft survival. However, Jessup et al.⁷⁷ used adenovirus to deliver an anti-CD4 single-chain, variable-domain antibody fragment (anti-CD4 scFvs) and found that, although these antibody fragments blocked allostimulation, their intraocular expression did not prolong corneal allograft survival, suggesting the possibility of tachyphylaxis.

Nerve growth factor (NGF) was reported to modulate an immune response from predominantly Th1 type to a Th2 type with pleiotropic effects on corneal wound healing.⁹⁶,⁹⁷ NGF gene therapy has been investigated for a variety of diseases. In a corneal allograft transplant model, NGF transfection using adenovirus resulted in improved graft survival and reduced inflammatory markers.⁷⁵ The authors also found that NGF and cytotoxic T-lymphocyte antigen 4 (CTLA-4)-Ig synergistically reduced apoptosis of corneal endothelium.

CTLA-4 is expressed on the surface of T helper cells, participating in the CD28–CD80–CD86 T-lymphocyte costimulatory pathway. CTLA4-Ig is a recombinant form of CTLA4 that acts as a competitive inhibitor of this CD28–CD80–CD86 pathway. Gene-based administration of CTLA4-Ig has shown promise in prolonging allograft survival in animal models.⁷³,^107–8 Comer and colleagues⁷³ described suppression of CD80 and CD28 expression using adenovirus-mediated delivery of CD80 mRNA into Brown Norway rat corneas, which were subsequently grafted into Lewis rat recipients. CD80 gene therapy was found to prolong graft survival. As expected, systemic administration produced better allograft survival. Using a ballistic transfer technique with plasmid-coated gold microparticles, Konig et al.⁸⁴ found that IL-4 and CTLA4 prolong corneal graft survival in comparison with the gold-treated control group and the IL-10-treated group. However, overexpression of IL-4 was not sufficient to reduce the rejection rate of corneal allografts in an experimental keratoplasty model of MHC class I/II-incompatible rats.⁸³ Ignatius et al.⁸⁵ reported increased survival of orthotopic corneal allograft in mice after ballistic delivery of IL-4/CTLA-4 plasmid-coated gold microparticles into the skin of the lower lid or shank.

Recent data suggest that Th17 and its products, e.g. IL-17, IL-21 and IL-22, may play a role in transplant immunity; however, this role is controversial. IL-17 is a proinflammatory cytokine that has been implicated in a variety of autoimmune diseases, e.g. multiple sclerosis, and allograft rejection of multiple tissues and organs in some studies. In vitro differentiation of the murine Th17 cell linage is controlled by intricate interactions between the cytokines and growth factors. CD28/B7-costimulation was found to downregulate Th17 development, whereas CTLA4-Ig, which interrupts the CD28/B7 pathway, favors Th17 differentiation. Antonysamy et al.⁹⁹ reported that IL-17 promotes differentiation of dendritic cells and up-regulates costimulatory molecules; IL-17 blockage using recombinant murine IL-17R:Fc fusion protein inhibits in vitro T cell proliferation and prolongs cardiac allograft survival. However, Gorbacheva et al.¹⁰⁰ reported that IL-17 promotes early allograft inflammation and tissue injury by facilitating T-cell recruitment into the graft. Recently, Chen et al.¹⁰¹ showed that neutralization of IL-17 using intraperitoneal injection of anti-IL-17 monoclonal antibody decreases angiogenesis and inflammatory cellular infiltration and improves allogeneic corneal graft survival. But, Yamada et al.¹⁰² used knockout mice (C57BL/6) to investigate the roles of IL-17 and INF-γ and concluded that neither INF-γ nor IL-17 played a critical role in the development of minor-specific allograft rejection in MHC disparate allograft transplantation. Another study reported that IL-17A regulates VEGF-A and VEGFR-1 expression and promotes corneal angiogenesis after HSV infection.¹⁰³ More recently, Cunnusamy and colleagues^104–6 found that IL-17A blocking, in mouse orthotopic keratoplasty model, actually increased the incidence of allograft rejection. The authors proposed that CD4+CD25+ regulatory T cells require IL-17A to mediate contact-dependent and time-dependent suppression.¹⁰⁶ Further investigations to unravel the roles of Th17/IL-17 in graft rejection are underway.

Indoleamine 2,3-dioxygenase (IDO) is an intracellular enzyme that induces tryptophan depletion, arrests activated T lymphocytes in the G1 phase, and interferes T cell-mediated allograft rejection. It has also been proposed that IDO mediates CTLA4Ig-induced tolerance via reverse B7 signaling in dendritic cells, leading to T-cell suppression.^109–10 Beutelspacher et al.⁷⁸ presented data showing that transfection of IDO to mouse cornea using equine infectious anemia virus produces overexpression of IDO and prolongation of allograft survival. IDO overexpression has been reported to improve allograft survivals in other solid organ transplant models.¹¹¹ Interestingly, cigarette smoke exposure appears to reduce long-term allograft survival by suppressing IDO expression.^112–113

CONCLUSION

The current literature suggests that significant and exciting progress has been made in the field of gene therapy for ocular diseases and, specifically, in experimental models for treatment of allograft rejection. The unique properties of the anterior chamber and the cornea facilitate laboratory research to elucidate the intricate molecular and cellular mechanism of allograft rejection and to identify targets for gene therapy. The diversity of cornea models allows the formulation of critical and clinically relevant questions and subsequent hypothesis testing. Current lines of investigation raise the possibility of gene-based prevention and treatment of allograft rejection in the near future.