Ординатура / Офтальмология / Английские материалы / Ocular Periphery and Disorders_Dartt, Bex, Amore_2011

Further Reading

Camper, S. A., Saunders, T. L., Kendall, S. K., et al. (1995). Implementing transgenic and embryonic stem cell technology to study gene expression, cell–cell interactions and gene function.

Biology of Reproduction 52: 246–257.

Chikama, T., Hayashi, Y., Liu, C. Y., et al. (2005). Characterization of tetracycline-inducible bitransgenic Krt12rtTA/+/tet-O-LacZ Mice.

Investigative Ophthalmology and Visual Science 46: 1966–1972. Funderburgh, J. L., Corpuz, L. M., Roth, M. R., et al. (1997). Mimecan,

the 25-kDa corneal keratan sulfate proteoglycan, is a product of the gene producing osteoglycin. Journal of Biological Chemistry 272: 28089–28095.

Hanks, M., Wurst, W., Anson-Cartwright, L., Auerbach, A. B., and Joyner, A. L. (1995). Rescue of the En-1 mutant phenotype by replacement of En-1 with En-2. Science 269: 679–682.

Hayashi, Y., Liu, C. Y., Jester, J. J., et al. (2005). Excess biglycan causes eyelid malformation by perturbing muscle development and TGF-alpha signaling. Developmental Biology 277: 222–234.

Kao, W. W. (2006). Ocular surface tissue morphogenesis in normal and disease states revealed by genetically modified mice. Cornea 25 (supplement 1): S7–S19.

Kao, W. W. and Liu, C.-Y. (2003). The use of transgenic and knock-out mice in the investigation of ocular surface cell biology. The Ocular Surface 1: 5–19.

Kao, W. W., Xia, Y., Liu, C. Y., and Saika, S. (2008). Signaling pathways in morphogenesis of cornea and eyelid. The Ocular Surface 6: 9–23.

Liu, C. Y., Shiraishi, A., Kao, C. W., et al. (1998). The cloning of mouse keratocan cDNA and genomic DNA and the characterization of its expression during eye development. Journal of Biological Chemistry 273: 22584–22588.

Muller, U. (1999). Ten years of gene targeting: Targeted mouse mutants, from vector design to phenotype analysis. Mechanisms of Development 82: 3–21.

Saika, S., Okada, Y., Miyamoto, T., et al. (2004). Role of p38 MAP kinase in regulation of cell migration and proliferation in healing corneal epithelium. Investigative Ophthalmology and Visual Science

45: 100–109.

Weng, D. Y., Zhang, Y., Hayashi, Y., et al. (2008). Promiscuous recombination of LoxP alleles during gametogenesis in cornea Cre driver mice. Molecular Vision 14: 562–571.

Xia, Y. and Kao, W. W. (2004). The signaling pathways in tissue morphogenesis: A lesson from mice with eye-open at birth phenotype. Biochemical Pharmacology 68: 997–1001.

Zhang, L., Wang, W., Hayashi, Y., et al. (2003). A role for MEK kinase 1 in TGF-beta/activin-induced epithelium movement and embryonic eyelid closure. EMBO Journal 22: 4443–4454.

Glossary

Alloantigen – An antigen that is the part of an animal’s self-recognition system. The common alloantigens are the major histocompatibility complex and red blood cells.

Astigmatism – The inability of the eye to focus sharp image on the retina due to the irregular shape of the cornea or lens.

Capsid – The protein shell that surrounds genetic material of the virus.

Corneal graft or penetrating keratoplasty – The replacement of the central diseased cornea with a healthy donor cornea. This process is also called corneal transplantation.

Corneal neovascularization – The ingrowth of new blood vessels from limbal plexuses toward clear cornea.

Corneal scarring or haze – The loss of corneal transparency and appearance of scar because of injury or abnormal wound healing.

Corneal stroma – The tissue between the epithelium and endothelium of the cornea. It constitutes approximately 90% of corneal thickness and comprises collagens, keratocytes, and extracellular matrix.

CRE-adenovirus vector – An adenovirus vector generated with the helper vector that has a packaging sequence flanked by Cre/lox-P system using human embryonic kidney 293 cells stably transfected with Cre recombinase. This results in selective deletion of packaging sequence from helper virus.

Dominant-negative mutant construct – A plasmid encoding for a mutant protein that competes with wild-type (normal) protein within the same cell to inhibit its function.

Enhanced green fluorescent protein (EGFP) –

A 27-kDa protein isolated from jelly fish. It fluoresces green when exposed to blue light and is commonly used as a marker for gene transfer studies. Hyperopia – A term used to define eye defect in which near objects appear blurred because images are focused on the back of the retina instead of on the retina. This medical condition is also known as faror long-sightedness.

Keratitis – The inflammation in the cornea causing pain and discomfort.

Myopia – A term used to define eye defect in which distant objects appear blurred because images are focused in front of the retina instead of on the retina. It is also called nearor short-sightedness.

Orthotopic graft/transplant – The transplantation or grafting of tissue at its normal position in the body. Reporter/marker gene – The gene(s) used to track and/or quantify levels of gene delivery in the cell. The common reporter genes are green fluorescent protein, beta galactosidase, etc.

Serotype – A characteristic set of antigens used to distinguish closely related virus strains. Sonoporation – A technique used to produce small pores temporarily in the cell membrane using ultrasonic waves for introducing RNA, DNA, or small drugs into the cells.

Vector – Virus or nonviral materials used as a vehicle for carrying genes into the cells.

Introduction

Gene therapy is an attractive approach to treat ocular surface diseases and disorders (Table 1). Recent reports of improved visual function in adult patients with Leber’s congenital amaurosis with gene therapy attest the potential of this form of molecular medicine to cure eye disease and prevent blindness. The front of the eye, commonly referred as ocular surface, primarily consists of the tear film, cornea, and conjunctiva. The lids, tears, and lacrimal glands are also considered integral parts of the ocular surface, as they spread tears and protect the eye. Nearly all ocular surface diseases entail an abnormality in the cornea. Consequently, more research has been performed to develop gene therapy approaches for the cornea compared to other ocular surface constituents. This article provides an overview of ocular surface gene transfer studies performed by us and many other investigators.

Corneal Gene Therapy Methods

Cornea is an attractive target for gene therapy because of its accessibility, immune-privileged status, and ability to be monitored visually. Gene transfer research in the

Health in Conjunctiva and Cornea Adnexa, Ocular Film, Tear the of Function and Structure 328

329 Gland Lacrimal and Conjunctiva, Cornea, the for Therapy Gene

cornea primarily focuses on developing therapeutic modalities for common clinical problems such as corneal neovascularization, graft rejection, corneal scarring, and wound healing. The scope of developing novel interventional gene therapy strategies has been markedly enhanced because of an increased understanding of the molecular mechanisms and pathogenesis of corneal diseases. Multiple vectors, techniques, and strategies have been utilized to deliver foreign genes in the cornea. Employing in vitro, ex vivo, and in vivo models, the efficiency of numerous viral and plasmid vectors to deliver genes in the ocular surface tissues has been evaluated. Among viral vectors, adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus vectors were found to efficiently introduce genes in the cornea. However, none of these vectors is ideal, and each had its own pros and cons.

Viral Vectors

Adenovirus and retrovirus vectors provided short-term transgene expression in the mouse and rabbit cornea with moderate-to-severe inflammatory response. These vectors also transduced corneal and conjunctival epithelium efficiently in human cornea but failed to deliver genes in rat, rabbit, and sheep corneal epithelium. Interestingly, adenovirus vector showed the best transduction efficiency for corneal endothelial cells when compared to the vectors developed from equine immunodeficiency virus, lentivirus, or bacculovirus. Nonetheless, both adenovirus and retrovirus vectors are of limited use for corneal gene therapy because of their inability to transduce nondividing cells, low transduction efficiency for corneal cells, and propensity to induce immune reactions. AAV and disabled lentivirus vectors can transduce nondividing cells and provide long-term transgene expression, thus offering good alternatives for delivering genes into keratocytes and endothelium of the cornea. However, limited studies have been performed to evaluate the utility of these vectors for corneal gene therapy. We, for the first time, showed selective transgene delivery into keratocytes of the rabbit cornea in vivo with AAV2 vector using a lamellar flap technique. Figure 1 shows the levels

and locations of b-galactosidase (b-gal) reporter gene expression in the rabbit cornea. This study also revealed that direct contact of vector to stroma is critical for

2 and 5 showed that AAV vectors provide long-term transgene expression in the mouse and rabbit stroma in vivo. The mouse eyes continued to express enhanced green fluorescent protein (EGFP) in the corneal stroma for 10 months, without showing any significant side effects (Figure 2).

In recent years, several hybrid AAV vectors have been engineered using the genome of AAV serotype 2 and capsid protein of AAV serotypes 1–9 for gene therapy. The newly produced hybrid AAV vectors have shown improved trangene delivery in many tissues including the eye. However, besides the AAV2/5 vector, transduction efficiency of other newly developed hybrid AAV vectors for the cornea has not been investigated. Recently, our laboratory tested the transduction efficiency of AAV2/6, AAV2/8, and AAV2/9 vectors for the human corneal fibroblasts and epithelial cells in vitro and for mouse cornea in vivo. The results showed that the tested hybrid AAV vectors are more efficient when compared to nonhybrid AAV2 serotypes for each corneal cell type. Figure 3 demonstrates the levels of transgene delivery in human corneal epithelium (a) and human corneal fibroblast (b) delivered with AAV2/6 vector. The analysis of AAV2/6, AAV2/8, and AAV2/9 gene transfer data revealed that AAV2/6 vector is the most efficient among the three tested vectors for human corneal fibroblasts and epithelial cells in vitro. Although AAV vectors are efficient and safe for delivering genes in the cornea, unfortunately they are incapable of transporting large genes (>1.8 kb).

Disabled lentivirus vectors have been used to deliver large genes to all three major cells of the human cornea in vitro and to mouse corneal endothelium in vivo. In addition, transgene delivered with lentivirus vectors showed long-term expression. Our research team noted efficient EGFP gene delivery into keratocytes of the mouse cornea in vivo with lentivirus vector that was applied to the mouse stroma for 2 min after removal of

epithelium. Figure 4 shows the quantity and distribution of transgene in the mouse cornea with lentivirus vector administered in the stroma topically or through microinjection technique. Detection of distinctly different levels and locations of transgene in the cornea supports our hypothesis that gene delivery in the cornea is regulated by both vector and vector-delivery techniques and not by the vector alone. No serious cytotoxicity or side effects have been reported with the AAVor lentivirus-mediated gene delivery, suggesting that these vectors may be suitable

for gene therapy treatments for patients. However, safety remains a major concern for using lentivirus vectors because of their origin from human immunodeficiency virus.

Nonviral Vectors

Contrary to viral vectors, nonviral vectors are nontoxic, nonpathogenic, and nonimmunogenic, and can deliver large genes. Additionally, their production is simple and

cost effective. Various surgical, mechanical, electrical, or chemical approaches were used to administer plasmid DNA in the cornea in vitro, ex vivo, and in vivo. Intrastromal injection of plasmid DNA encoding reporter, vascular endothelial growth factor, or interleukin-1 receptor antagonist successfully delivered transgene into the mouse cornea. A major advantage of this method over viral technology was that it provided rapid transgene expression in the mouse cornea in vivo. Transgene expression was detected as early as 1h after administration, with a peak occurring in 12–24 h. Furthermore, the plasmid-injected corneas remained clear and free of inflammation. Plasmid applied during stromal hydration and filtration bleb surgeries delivered genes in rabbit corneas, in vivo.

Techniques using electric current plasmid DNA delivery in corneal epithelium, keratocytes, and endothelium have been reported. Electrical current up to 200 V cm 1 did not cause trauma, edema, or inflammation in the cornea but introduced only low levels of transgene. Higher electrical current increased transgene delivery but was associated with substantial corneal damage.

Firing of DNAor RNA-coated gold microparticles on the cornea with a gene gun introduced transgene in the corneal epithelium. However, gene-gun-mediated gene delivery was limited to epithelium and was associated with corneal damage.

Lipids and polymers have been used frequently to deliver genes in corneal cells in vitro but provide inadequate levels of transgene in the cornea in vivo. Low transfection efficiency and short-term transgene expression are among the major challenges of nonviral gene therapy that need to be addressed. Use of nanoparticles as vectors has great potential for improving nonviral gene transfer.

Use of Gene Therapy to Treat Corneal Diseases

Corneal Graft Rejection

Many gene therapy studies for prevention of corneal graft rejection have focused on the use of cytotoxic T- lymphocyte-associated antigen-4 conjugated to human immunoglobulin G (IgG) heavy chain (CTLA-4 Ig). The rationale for this therapy is based on the fact that helper T-cell activation primarily involves the interaction between T-cell receptor/cluster of differentiation 3 (CD3) and the alloantigen/major histocompatibility complex II (MHCII) of the antigen-presenting cells (APCs). An additional, essential stimulus for T-cell activation is provided by the interaction between co-stimulatory molecules, such as APC B7 antigens, and the CD28 molecule of T cells. T-cell activation can be prevented by blocking this interaction with administration of chimeric protein, CTLA-4 Ig. Multiple studies have evaluated the efficiency of CTLA-4 Ig gene therapy to control corneal graft rejection.

The CTLA-4 Ig gene therapy was delivered to the donor rat corneas with adenovirus prior to transplantation topically or after transplantation by a single intravenous or intraperitoneal injection. Topical application of adenovirus expressing CTLA-4 Ig had a marginal effect on preventing graft rejection, whereas systemic administration markedly prolonged graft survival. In another study, minimalistic immunologically defined gene expression (MIDGE) vector encoding for CTLA-4 was delivered in the corneal epithelial cells with a gene gun 10 days after orthotopic corneal transplantation. A marginal beneficial effect of CTLA-4 expression was noted for graft survival in the tested model. However, a subsequent study of MIDGEmediated CTLA-4 gene transfer in mice demonstrated significant prolongation of graft survival when gene therapy was delivered 1 day before corneal transplantation. Contrary to the encouraging CTLA-4 gene transfer studies, the results have been disappointing when other costimulatory pathways of T-cell activation are inhibited. Adenovirus-mediated ICOS, which is an inducible costimulatory receptor expressed by activated T cells, ex vivo or systemic gene therapy did not result in a significant prolongation of corneal graft survival.

Cytokines play an important role in corneal graft survival and rejection. Consistently elevated levels of proinflammatory cytokines, such as Th (T-helper cell) type 1, have been detected in the corneal tissues and aqueous humor of eyes with graft rejection. Th type 2 cytokines, such as interleukin (IL)-10 and IL-4, exert inhibitory effects on Th1 cytokines. The effects of elevation of anti-inflammatory Th type 2 cytokines or inhibition of proinflammatory Th type 1 cytokines on graft survival have been the focus of many gene therapy studies. Significantly increased corneal graft survival, from 18–20 days to 45–55 days, was observed in ovine donor corneas transduced with adenovirus-expressing IL-10 or IL-12 antagonist. On the other hand, adenovirus-mediated IL-10 or IL-12 antagonist gene therapy did not prevent corneal transplant rejection in a rat model. This disparity in results could be due to differences in the kinetics of graft rejection in the two animal species. Interestingly, IL-4 gene therapy was found to be ineffective in both the species.

Corneal Wound Healing

Wound healing plays important role in maintaining corneal transparency and normal visual function. Injury to the cornea is known to trigger unregulated woundhealing response that can lead to scar formation and loss of vision. Multiple growth factors and cytokines have been shown to regulate wound healing in the cornea. Out of many cytokines influencing corneal wound healing, transforming growth factor beta (TGFb) has been shown to play a central role in corneal wound healing, myofibroblast generation, and haze development. Soluble

type II TGFb receptor binds TGFb and blocks its biological activity. Soluble type II TGFb receptor gene therapy has been shown to reduce corneal opacification in corneal injury model. A single intramuscular injection of recombinant adenovirus encoding for soluble type II TGFb receptor in the mouse eye prevented corneal edema, angiogenesis, inflammatory cell infiltration, and deposition of extracellular matrix in the cornea. High levels of soluble TGFb receptor were detected in the serum and corneal fluid of treated animals for 10 days but several side effects were also observed. This study demonstrated the potential application of gene therapy to treat corneal disorders but several issues such as safety, immunological reaction, etc., associated with adenovirusbased gene therapy still need to be resolved.

AAV-mediated gene therapy may address some of these issues. Our laboratory, for the first time, demonstrated efficient transgene delivery into keratocytes of the normal and diseased (hazy and neovascularized) rabbit corneas in vivo with AAV2 and AAV5 vectors. The tested AAV vectors showed high levels and long-term transgene expression (over 4 months) in the rabbit corneas. Our ongoing gene transfer experiments include evaluation of AAVmediated decorin gene therapy to control corneal scarring and angiogenesis. Decorin is a small leucine-rich proteoglycan and blocks biological activity of TGFb. Our in vitro studies revealed that decorin gene delivery into rabbit and human keratocytes significantly reduces TGFb-driven transdifferentiation of keratocyte to myofibroblasts. This transformation is believed to cause corneal haze in vivo.

Employing nonviral gene transfer approach, the role of fibrin deposition on corneal transparency was also studied in laser-induced fibrin clot formation model in vivo. Administration of plasmid-DNA-expressing tissue plasminogen activator gene through electroporation in the anterior chamber of the eye markedly reduced extracellular matrix deposition in treated eyes. The beneficial effects lasted for 4 days.

Corneal Alkali Burn

Alkali burn injury to the cornea is a serious clinical problem that often leads to permanent visual loss due to ulceration, scarring, and/or neovascularization. A gene therapy approach has been used to treat corneal alkali burn by targeting modulation of TGFb super family genes using adenovirus vectors. Mothers against decapentaplegic homolog 7 (Smad7) is well documented to inhibit TGFb signaling. Topical application of Cre-adenovirus encoding for Smad7 effectively prevented alkali-induced corneal scaring. Bone morphogenic protein-7 (BMP7) is another member of TGFb super family and has been shown to antagonize the effects of TGFb. Adenovirusmediated BMP7gene transfer in the cornea has been

shown to accelerate re-epithelilization of corneal surface and suppress myofibroblast generation, monocytes/macrophages infiltration and macrophage chemoattractant protein-1 (MCP-1), and TGFb and collagen expression in the corneal stroma in alkali-induced corneal injury mouse model. However, BMP7 gene therapy was ineffective to suppress stromal neovascularization. Another study reported that topical application of Cre-adenoviral vector encoding for peroxisome proliferator-activated receptor-gamma (PPARg) reduced inflammatory and fibrogenic responses in the alkali burn mouse cornea. Adenovirus-mediated PPARg overexpression inhibited growth factors and upregulation of matrix metalloproteinases, monocytes/macrophages infiltration, and myofibroblasts production in healing cornea in vivo. Further, accelerated re-epithelization and basement membrane reconstruction was observed in mouse cornea overexpressing PPARg gene.

Corneal Scarring or Haze

Corneal scarring is primarily caused by injury or infections to the eye. It is also a common side effect of the photorefractive keratectomy (PRK) surgery, particularly in patients undergoing high myopia correction. The PRK surgery is frequently used worldwide to treat myopia, hyperopia, and astigmatism. Accumulating evidences suggest that corneal scarring occur due to abnormal wound healing, deposition of disorganized extracellular matrix, keratocyte activation, and transdifferentiation of keratocytes to fibroblasts and myofibroblasts. Gene therapy offers a unique approach for intercepting the molecular events involved in the development of corneal haze. Blockade of keratocyte proliferation is an attractive approach for controlling corneal haze in vivo. Rabbit corneas transduced with retroviral vector encoding for herpes simplex virus (HSV) thymidine kinase gene after keratectomy, followed by topical application of ganciclovir, showed significant inhibition of laser-induced corneal haze. Another approach that has been tested to control corneal haze is the blockade of cyclins and cyclin-dependent kinases that play an active role in controlling cell division. Again using a retroviral vector, a dominant-negative mutant construct for cyclin G1 was delivered in the rabbit cornea after transepithelial phototherapeutic keratectomy surgery. Analysis of corneal tissues showed that dominant-negative cyclin G1 delivery decreased extracellular matrix production and markedly reduced the development of corneal haze in tested rabbit model. Apoptosis of activated keratocytes by the delivered dominant-negative cyclin is believed to be a mechanism for the decrease in corneal haze appearance. As noted above, we have also shown the potential of decorin gene therapy to inhibit corneal haze using an in vitro model.

Corneal Neovascularization

Corneal insults, such as infection, degenerative disease, chemical damage, mechanical/surgical injury, and immunologic disease, can cause corneal neovascularization (CNV) that affects about 1.4 million Americans each year. Multiple lines of evidence suggest that cytokine vascular endothelial growth factor (VEGF) plays a key role in CNV development. Various gene therapy strategies have been developed and examined to suppress VEGF-induced corneal angiogenesis using experimental models. Intrastromal injection of plasmid encoding a soluble form of the VEGF receptors Flt-1, which neutralizes VEGF, significantly inhibited CNV in the mouse eye. Similar inhibition of CNV was demonstrated by sFlt1 gene transfer with adenovirus and AAV. In a novel approach for controlling CNV with gene therapy, plasmids encoding for Flt23K or Flt24K peptide (VEGF-binding domains of sFlt-1) coupled with endoplasmic reticulum-retaining peptide have been developed. These peptides are not secreted outside the cell and therefore are capable of neutralizing intracellular VEGF. Intrastromal injection of these plasmids in mouse eye inhibited CNV by as much as 50% in the experimental models of corneal angiogenesis. Recently, these scientists reported CNV reduction up to 40% in the mouse cornea after delivering Flt23K gene with albumin nanoparticles.

Gene therapy has also been evaluated in a corneal transplant rabbit model to study the effect of gene transfer of a fusion protein. These rabbit corneas were transduced ex vivo with lentiviral vector encoding for endostatin and kringle-5 domain of plasminogen fusion protein. Endostatin is an anti-angiogenic peptide derived from type XVIII collagen, and Kringle 5 domain of plasminogen is a specific inhibitor for endothelial cell proliferation derived from the proteolytic fragment of human plasminogen. The endostatin-kringle-5 fusion protein effectively inhibited allogenic transplantation-induced neovascularization. Delivering kringle-5-plasminogen gene through electroporation was also demonstrated to be effective in reducing CNV in a rat model. Angiostatin is another potent, recently identified anti-angiogenic agent. AAV vectors encoding angiostatin gene effectively reduced alkali-induced CNV in rat corneas. In another study, mouse corneas transfected with plasmid encoding for IL12 or IL10, using topical application or intrastromal techniques, showed marked suppression of CNV. Interestingly, a careful review of the literature reveals that few gene transfer studies of CNV in experimental disease models have reported, discussed, or investigated the side effects or downsides of the gene transfer methods that were investigated.

Corneal Dystrophies

In the past decade, several genes and gene mutations, such as BIGH3, TGFb1, gelsolin, and CHTS6 have been identified to cause granular, lattice, Avellino, and/or

Reis–Bu¨cklers corneal dystrophies. However, a paucity of experimental models for evaluating the usefulness of gene therapy to cure genetic corneal dystrophies remains a major obstacle to the development of therapeutic interventions for these vision-threatening disorders. Very few gene transfer studies using corneal buttons collected from patients or rodent models have been performed to evaluate the efficacy of gene therapy in corneal dystrophies. Gene therapy approaches can be used as a tool for creating animal models of corneal dystrophies by overexpressing aberrant proteins in the cornea.

Mucopolysaccharidosis is a group of metabolic disorders characterized by deficiency of lysosomal enzymes needed to degrade glycosaminoglycans. This disorder causes corneal abnormalities, including the loss of corneal transparency. Delivering human beta-glucuronidase gene in the cornea induced significant clearance of corneal clouding in mouse model of type VII mucopolysaccharidosis. The adenovirus vector encoding human betaglucuronidase gene was injected in the anterior chamber or intrastromal region to deliver transgene in the eye. A rapid clearance of lysososmal-storage vesicles in keratocytes and clearing of the cornea were observed.

Other Corneal Disorders

HSV keratitis is a leading cause of infectious blindness. Developing vaccination using gene transfer approaches have been the focus of several studies. Most of these studies used plasmid DNA encoding for HSV-1 glycoproteins, such as glycoprotein (g) D, gB1, or a cocktail of glycoproteins in an effort to increase immunity against HSV-induced ocular keratitis. The plasmids were administered in test animals through either topical application, or subconjunctival, intramuscular, or intraperitoneal injection. The intramuscular route of administration has been found to confer total protection against HSV keratitis, whereas topical application and subconjunctival administration prevented stromal keratitis but not the epithelial keratitis. In another study, enhancement of gD glycoprotein was observed when it was administered in combination with IL-2. Conversely, many investigators have reported limited success in controlling herpes keratitis with gene therapy, as a topical application of naked plasmid vector encoding for cytokines, such as IL2, IL4, IL10, interferon (IFN), or tumor necrosis factor (TNF) alpha, demonstrated very little or no benefit. Nonetheless, cytokine gene therapy has been shown to inhibit corneal lesion severity if used 3 days prior to HSV infection. Clinically, such treatment may be beneficial for preventing primary HSV keratitis.

Lacrimal Gland Gene Therapy

Lacrimal glands produce tears and tear proteins which lubricate, and supply nutrition and protection to ocular

surface tissues. Several autoimmue disorders such as Sjo¨gren syndrome, rheumatoid arthritis, systemic lupus erythematosus, and uveitis affect lacrimal gland function and result in dry eye syndrome. This syndrome affects about 4% people in the United States. Unlike conventional therapy that demands repeated lubricant application, gene therapy could potentially require just one treatment. Genes delivered to lacrimal glands can modulate tear composition and flow rate. Initial studies tested the efficacy of vaccinia, herpes, and adenovirus vectors for delivering genes in lacrimal gland using ex vivo rat lacrimal gland. Vaccinia viral vector showed highest transgene delivery followed by adenovirus and herpes viral vector. Histological examination of tissues revealed that vaccinia vector delivered transgene in the lacrimal duct cells and acini, whereas adenovirus vector mainly transduced myoepithelial cells surrounding the lacrimal acini. Cellular degradation response was also noted with adenovirus vector, possibly due to vector’s toxicity. Subsequent studies showed IL-10 and TNF inhibitor gene delivery in cultured rabbit lacrimal gland epithelial cells by the adenovirus. In vivo studies examined the efficacy of these cytokines-based gene therapy for Sjo¨gren syndrome, using a rabbit model of dacryoadenitis. The prophylactic adenovirus-mediated IL-10 gene delivery resulted in protection against lacrimal gland immunopathology, ocular surface disease, and decrease in tear production. On the other hand, adenovirusmediated TNF inhibitor gene delivery increased basal tear production and its stability, and reduced corneal surface defects and intensity of immune cell infiltration in lacrimal gland.

Adenovirus-mediated gene delivery has also been utilized to investigate cellular and physiological functions of the lacrimal gland. Examples include investigation of androgens in the pathophysiology of Sjo¨gren syndrome and the role of dyneins, protein kinases, and cytoskeletal proteins in the secretory functions of the lacrimal gland. Besides adenovirus, not many other viral vectors have been evaluated for lacrimal gland gene delivery in vivo. Retroviral vectors expressing the E6 and E7 genes of the human papillomavirus have been employed to generate immortalized lacrimal gland epithelial cell lines for laboratory studies.

Conjunctiva Gene Therapy

A handful of gene transfer studies have been performed for the conjunctiva. Recently, transfection efficiency of hyaluronic acid–chitosan nanoparticles to deliver genes in human conjuctival cells has been examined. These nanoparticles were found to be nontoxic to the conjuctival cells and exhibited moderate transfection efficiency (15%) for the conjunctiva. Topical application of these nanoparticles to the rabbit eye demonstrated a successful

delivery of reporter gene into conjunctival epithelium. Sonoporation (application of sound waves), following subconjuctival injection in the eye, delivered considerable levels of EGFP in the rat conjunctiva.

Among viral vectors, adenovirus has been most widely investigated for transgene delivery in the conjunctiva. Modulation of wound healing and prevention of scarring in the conjunctiva has been the primary focus of conjunctival gene therapy studies. As in corneal injury, TGFb has been shown to play a critical role in conjunctiva wound healing and scarring. Activation of p38 mitogen-activated protein (MAP) kinase is one of the key signaling pathways activated by TGFb. Adenovirus-mediated delivery of p38 MAPkinase suppressed myofibroblast generation and decreased messenger RNA (mRNA) expression of connective tissue growth factor (CTGF) and monocyte/ macrophage chemoattractant protein-1 (MCP-1) in the mouse model of conjunctival injury. The protective effects of Smad7 gene transfer on conjunctival fibrosis have also been reported using a mouse model. Cultured subconjunctival fibroblasts transduced with adenoviral vector expressing Smad7 inhibited type-I collagen, a smooth muscle actin, and CTGF, whereas topical application of adenoviral vector expressing Smad7 gene prevented macrophage invasion and decreased VEGF and a smooth muscle actin levels in conjunctival fibroblasts in vivo. More recently, adenovirus-mediated PPARg gene transfer was found to be protective against conjunctival fibrosis. PPARg overexpression suppressed expression of type I collagen, fibronectin, and CTGF in cultured human conjunctival fibroblasts both at mRNA or protein level. In vivo experiments showed that PPARg gene delivery significantly decreased monocyte/macrophage invasion, myofibroblast generation, and blocked upregulation of cytokines/growth factors, collagen I, and a2 mRNA in the healing conjunctiva.

In summary, many studies have demonstrated the ability of gene therapy to treat ocular surface diseases and improve visual function. Numerous vectors, techniques, and strategies have been identified for establishing novel gene therapy modalities to treat ocular surface disorders. Although the proof-of-principle experiments validated the potential and promise of gene therapy for ocular surface disorders, several obstacles remain to be cleared before gene therapy clinical trials for these diseases are instituted.

Acknowledgments

The work is supported by the RO1EY17294 (RRM) grant from the National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA and a grant from the Research to Prevent Blindness, New York, USA.