Prospects for gene therapy in corneal disease

ABSTRACT Transfer of cDNA to corneal cells has been accomplished using viral and nonviral vectors. Studies examining the feasibility and optimal methods for vector-mediated gene transfer to

the cornea have, as in other tissues, been performed using histochemical or fluorescent marker genes. These have used corneal cells or cell lines _in vitro_, and whole corneas maintained in

_ex vivo_ culture. Gene-based interventions have been examined in specific corneal disorders such as allograft rejection, postexcimer laser scarring, and herpes simplex keratitis using

experimental models. As the feasibility of genetic modification of corneal cells has been successfully demonstrated, there is great potential for gene therapy vectors in the treatment of

human corneal disease. Continued improvements in vectors for gene transfer will improve the efficacy and safety of gene therapy. In addition to use of cDNA transfer as an alternative to drug

or protein treatments in acquired corneal disorders, our expanding knowledge of the genetic basis of inherited corneal disorders will ultimately lead to the development of specific and

effective gene therapies in this category of diseases. INTRODUCTION A number of characteristics of the cornea compared with other tissues confer significant potential for gene treatment of

corneal diseases. These include the relatively simple histological structure of the cornea, its accessibility for examination and manipulation, its ability to be maintained in _ex vivo_

culture for several weeks, and the relative immune privilege of the anterior chamber.1 Ongoing advances in our understanding of the biological basis of corneal diseases such as the inherited

epithelial, stromal, and endothelial dystrophies,2,3 allograft rejection,4 herpes simplex keratitis,5 and others provide a growing number of possible targets for the development of genetic

therapies. Corneal diseases tend to affect primarily a single layer of tissue, and gene therapy approaches for specific diseases will most likely require modification of the primary site of

pathology. For example, the corneal epithelium is rapidly dividing and is readily accessible to topical agents. The stroma and keratocytes are physically sequestered by the overlying

epithelium/Bowman's layer and the lamellar structure of the extracellular matrix. The endothelium is a nonreplicative monolayer, directly accessible to interventions _in vivo_ via the

anterior chamber and _ex vivo_ in whole corneal storage. Numerous _in vitro_ and nonhuman _in vivo_ studies reported to date have established the feasibility of delivery modalities for each

layer of the cornea. Furthermore, studies in animal models have shown that the exogenous administration of specific genes can inhibit pathological processes affecting different corneal

layers. These results collectively emphasize the significant potential of these approaches. TECHNIQUES FOR GENE TRANSFER TO THE CORNEA EPITHELIUM The accessibility of the corneal epithelium

is a clear practical advantage for potential gene therapy applications. Furthermore, the central role of epithelial cells in the well-defined keratoepithelin dystrophies makes this layer an

appealing target for genetic modification.6 Earliest attempts to genetically modify corneal epithelium used the physical method of bombardment with gold microparticles coated with DNA

encoding green fluorescent protein (GFP) marker. This approach successfully delivered the marker gene to the target cells without corneal damage or ocular irritation.7 There has been greater

interest in use of recombinant viral vectors for gene transfer, principally on account of the ability of viruses to generate high levels of transgene expression. A replication-deficient

adenovirus type 5 (Ad5) bearing _lacZ_ cDNA encoding β-galactosidase was used by Tsubota _et al_8 to infect corneal epithelial cell lines as well as _ex vivo_ corneal epithelial cells with

100% transduction efficiency, meaning that all target epithelial cells were transduced. Application of the same Ad5 construct failed to yield transgene expression _in vivo_ in rat corneal

epithelium8 and _ex vivo_ to rabbit corneal epithelium.9 Upregulation of the pro-inflammatory cytokines interleukin-6, -8, and ICAM-1 was observed in response to Ad5 infection.8 This

upregulation was inhibited with coadministration of topical betamethasone, suggesting a possible approach to improve the usefulness of adenovirus-mediated gene transfer to the ocular surface

epithelium. STROMA In an alternative approach, the commonly used surgical technique of stromal hydration was used to inject a saline solution containing naked plasmid DNA directly into

mouse corneal stroma.10 This method demonstrated expression of _lacZ_ in epithelial cells and keratocytes at the remarkably early interval of 1 h postinjection, peak expression levels at 24 

h, and minimal inflammatory reaction. Use of this technique with a plasmid encoding vascular endothelial growth factor (VEGF) resulted in marked corneal vascularization. Similar experiments

with a plasmid encoding the VEGF inhibitor Flt-1 showed significant inhibition of the vascularization induced by controlled-release VEGF pellets implanted into mouse corneas.10 Genetic

modification of keratocytes has received particular attention in efforts to inhibit haze formation following refractive laser stromal ablation. A G1BgSvNa retroviral vector containing herpes

simplex virus (HSV) thymidine kinase (HStk) resulted in 90% growth inhibition of cultured human keratocytes.11 Following superficial keratectomy of rabbit corneas _in vivo_, the same vector

bearing _lacZ_ yielded 25–40% keratocyte transduction efficiency; treatment of HStk-transduced keratocytes with ganciclovir resulted in a statistically significant reduction in corneal

haze.11 Inhibition of rabbit keratocyte growth _in vitro_ has also been demonstrated using a pG1XSvNA retroviral vector containing an antisense cyclin G1 (aG1) construct by McDonnell and

colleagues.12 Transduction efficiency was 34% and cell proliferation was inhibited by approximately 50%. Transduction with aG1 was associated with increased incidence of apoptosis. This

study was extended to inhibit stromal haze after excimer laser photorefractive keratectomy (PRK) in rabbit eyes.13 Retroviral vector containing an antiproliferative dominant negative mutant

cyclin G1 (dnG1) was applied as drops after PRK. The dnG1 construct demonstrated significant inhibition of corneal haze at 2 weeks post-PRK. Later timepoints were not reported.13 Long-term

genetic modification of keratocytes may be a useful therapy for keratoconus and inherited stromal disorders. Mucopolysaccharidosis VII (MPS VII) is one such disorder that has been

investigated, an autosomal recessive disorder arising from systemic deficiency of lysosomal β-glucuronidase (GUSB) and causes ocular abnormalities including corneal clouding, retinal

degeneration, and glaucoma. Using a mouse model of MPS VII with corneal clouding, an adenoviral vector encoding GUSB was applied directly to corneal stroma after lamellar keratectomy. This

resulted in widespread distribution of GUSB expression in cells in the corneal stroma, epithelium, and endothelium at 5 and 30 days post-treatment.14 Corneal expression of GUSB was

associated with morphologic improvement as well as rapid and almost complete reversal of vacuolar histopathological changes. Even taking into account the shorter lifespan of mouse than man,

these results must be considered short-term for an inherited disorder in which lifelong expression of a functionally active therapeutic gene would be necessary. As long-term expression of a

therapeutic gene requires integration into the host cell genome, retroviruses are at present the vectors of choice for these applications. Lentivirus is one such vector and has been found to

transduce primary keratocyte cultures with high efficiency and sustained expression of GFP up to 60 days.15 Efficient gene transfer using the same retroviral vector was also reported _in

situ_ for endothelial and epithelial cells and for keratocytes at the cut edge of the specimen.15 As with other vectors, marker gene expression was not detected in keratocytes which were not

directly exposed to retrovirus-containing media. ENDOTHELIUM Following exposure of whole-thickness cornea _ex vivo_ to optimal concentrations of recombinant adenovirus bearing _lacZ_, high

proportions of rat (90%) and rabbit (>75%) endothelial cells were transduced. Expression was restricted to the endothelium, peaked at day 3–5 postinfection and was found to diminish to

very low levels at 14–21 days.9,16 Following adenovirus-mediated _lacZ_ transfer _ex vivo_, rabbit corneas were then transplanted as allografts into recipient rabbits. Short-term expression

of the marker gene in donor corneal endothelial cells was demonstrated _in vivo_, without significantly increased clinical or histopathological evidence of ocular inflammation. Moreover,

stable corneal thickness measurements postgrafting indicated that endothelial function remained satisfactory, excluding significant cytopathic effects of the virus or the tissue

manipulation.9 Further examining the feasibility of such approaches in clinical management, a similar adenovirus vector was shown to transduce human corneal endothelium _ex vivo_. High

(90–100%) levels of target cell transduction were found, with expression of the marker gene for a maximum of 7 days post-transduction.17 A similar vector was used to transfer cDNA encoding

CTLA-4 Ig, an immunomodulatory protein which blocks T-lymphocyte activation, into human corneas in _ex vivo_ culture: cumulative secretion of functionally active CTLA-4 Ig by the cornea into

culture medium was demonstrated up to 28 days post-transduction.17 The significance of this study is the demonstration that transfer of immunomodulatory genes, or other genes that might

influence graft function post-transplantation, to donor human corneas is feasible during the period of _ex vivo_ storage using current eye banking methods. In contrast to short-term

applications in such circumstances as transplantation, long-term genetic modification of corneal endothelial cells would be a significant therapeutic advance for endothelial disorders such

as Fuchs' dystrophy and inherited diseases. Integration of a putative therapeutic gene into the host cell genome would be required, for which candidate vectors are recombinant

adeno-associated virus (rAAV) or retrovirus. rAAV has been used _in vivo_ to transduce rabbit corneal endothelium after injection into the anterior chamber, although it is noteworthy that

highest level expression required induction of intraocular inflammation by intravitreal lipopolysaccharide injection.18 Nevertheless, evidence of _lacZ_ expression in endothelial cells up to

60 days postinjection suggests the feasibility of longer term genetic modification. Moreover, recombinant HIV-based lentivirus vectors delivered by a similar intracameral injection approach

have yielded gene expression in mouse endothelium for up to 12 weeks duration and without any requirement for induction of intraocular inflammation.19 The clear safety limitations for

clinical gene therapy applications in corneal disease of HIV-based retrovirus vectors has prompted investigation of non-HIV retroviruses. We have used an equine infectious anaemia virus

(EIAV) to transduce _ex vivo_ cultured human corneal endothelium (Figure 1 ). Transduction efficiency of this vector of approximately 50% suggests significant potential for nonhuman

retroviral vectors, which have the advantages of decreased pathogenicity and immune responses. Nonviral gene transfer vectors have several potential advantages over viral vectors. These

avoid the potential for viral cytopathogenicity and induced immunogenicity, and are comparatively easy to produce. Such vectors include cationic liposomes and cationic dendrimers.20,21,22

All of these studies showed that less than 10% of target cells were transfected with the marker gene, suggesting limited clinical potential of these agents alone. Receptor-targeting peptides

include integrin-targeting23 and transferring receptor-targeting24 vectors, nonviral systems that improve cell transfection efficiency rates in corneal endothelium to ∼25%. GENE THERAPIES

FOR CORNEAL DISORDERS CORNEAL GRAFT REJECTION Cornea is the most commonly transplanted tissue, with approximately 45 000 cases performed annually in the United States and the United Kingdom.

The fact that almost all donor corneas used as allografts in these procedures are stored _ex vivo_ for a period of up to 4 weeks prior to transplantation indicates the potential for genetic

modification approaches which side-step the possible safety issues of _in vivo_ gene therapy in patients. As graft rejection is the leading cause of corneal transplant failure, modulation

of the host allogeneic response to the cornea or protection of the donor corneal endothelium by some strategy has significant appeal. As in other transplanted tissues, corneal allograft

rejection is dependent on alloreactive T-cell activation.25 T-cell activation requires the initial interaction of alloantigen/major histocompatibility complex (MHC) present on the surface of

antigen-presenting cells (APC) with the T-cell receptor/CD3 complex present on the T-cell surface.25 T-cell activation requires additional costimulation interactions between molecules

expressed on the T-cell and APC surfaces. Binding of CD28 on the T-cell surface to ligands CD80 and 86 on the APC surface is the most potent such costimulatory interaction described thus

far. A strategy to prolong corneal graft survival by inhibiting the host T-cell costimulatory signal through CD28 has been examined in a rat corneal allograft model using the protein

CTLA4-Ig. The protein was expressed from cDNA in an adenoviral vector AdCTLA. These experiments demonstrated that _ex vivo_ administration of AdCTLA4 prior to transplantation minimally

prolonged graft survival, and protein would in this circumstance be assumed to be secreted into the anterior chamber by donor endothelial cells postgrafting; of interest, a single systemic

injection of AdCTLA at the time of transplantation significantly prolonged graft survival.26 An alternate immunomodulatory approach to prolong corneal allograft survival is inhibition of the

activity of tumor necrosis factor (TNF), a pro-inflammatory cytokine which is present in aqueous humour prior to and at observed onset of endothelial graft rejection.27 Using a rabbit

corneal transplant model, an adenoviral vector AdTNFR containing cDNA encoding a soluble TNF receptor protein was used to transfect donor corneas _ex vivo_ prior to transplantation.

Transduced corneas showed moderately increased graft survival times compared with control donor corneas incubated in virus-free medium. However, additional control corneas transduced with an

adenovirus construct lacking the TNF receptor (Ad0) showed significantly reduced graft survival times compared with AdTNFR-infected and mock-infected corneas. In tandem with the effects of

AdCTLA, this suggests harmful immunogenic effects of adenovirus infection which counteract the anti-inflammatory effect of the TNFR or CTLA-Ig constructs.28 In a related approach using

adenovirus and the TNF pathway, lipoadenofection was used to introduce a marker gene under a TNF-inducible promoter into rabbit corneal endothelium.29 This method utilizes adenovirus to

enhance liposome-mediated DNA transfer into cells and may have the potential advantages of decreased immunogenicity and no need for cloned adenoviral constructs. This study demonstrated a 9-

to 10-fold upregulation of marker gene expression after TNF stimulation, suggesting the possibility that endogenous TNF levels can be used to control the expression of additional

immunomodulatory genes to prolong graft survival.29 The significance is that it may be possible to commence expression of counter-inflammatory transgenes only at onset of a rejection

episode. An additional gene target for immunomodulation after corneal grafting is interleukin (IL)-10. This cytokine downregulates MHC class II and costimulatory molecule expression on

monocytes, macrophages, and dendritic cells, and inhibits the synthesis of pro-inflammatory cytokines. Using a sheep corneal transplant model, _ex vivo_ transfection of corneal endothelial

cells with an adenoviral construct encoding IL-10 demonstrated very significant prolongation of graft survival. IL-10 expression was detected at 21 days, and no evidence of immunologic or

inflammatory responses was present after transplantation.30 This study involved a similar _ex vivo_ donor cornea gene modification strategy and an adenovirus vector, but the more beneficial

effect on graft survival indicates the importance of cDNA selection in such studies. There is clear potential for improvement in effects of such gene modification strategies and this is

likely to follow improved less immunogenic viral or more effective nonviral vectors. Looking to possible application of these methods in the future, there is no reason why donor cornea

modification could not incorporate immunomodulatory with other cytoprotective constructs, and this would be likely to be combined with postoperative immunosuppression of the recipient.

HERPES SIMPLEX VIRUS KERATITIS It is known that after primary infection, HSV-1 assumes a latent state from which it can reactivate and cause corneal inflammation, vascularization, and

scarring. Detailed knowledge of the HSV genome and the immunological response following infection in host tissue is the foundation for recent studies on gene-based interventions in HSV

keratitis models. The host cytokine interferon-_α_1 (IFN-_α_1) antagonizes HSV-1 transcription, translation, and assembly. Using a murine model of lethal encephalitis after corneal infection

by HSV-1, topical application to the cornea of plasmid DNA encoding IFN-_α_1 provided a protective effect when administered either before or shortly after infection.31 Immunization confers

protection against viral infection. In an alternative approach to attenuating virus-induced injury, subconjunctival and topical administration of plasmid DNA encoding HSV-1 glycoprotein D

linked to interleukin-2 (gD-IL-2) prevented the development of keratitis in mice.32,33 These studies used murine models of primary HSV-1 infection and provide significant insights into the

pathobiology of HSV-1. They represent DNA vaccine approaches rather than gene therapy as discussed in other contexts. Human application of these approaches appears unlikely as widespread

pre- or peri-infection immunization with these vectors would face significant practical obstacles. CONCLUSIONS Genetic modification has been achieved in cultured corneal cells, _ex vivo_

corneas, and _in vivo_ animal models. Recombinant adenovirus vectors remain the most effective studied to date in the cornea, largely on account of the high proportion of target cells

transfected by these viruses and the resulting high levels of recombinant protein produced. Nonviral gene transfer systems have not as yet proved effective, although toxicity and

immunogenicity of viral vectors are likely to limit the safety of these in clinical applications. However, later-generation adenovirus vectors, in which deletion of more viral genes is

engineered, will be of experimental interest especially in immunological applications, in which any corneal cell expression of viral proteins can significantly attenuate the immunomodulatory

effect of the therapeutic gene. An additional major challenge in development of gene therapies in corneal disorders is the requirement of long-term, even lifelong, gene expression for

inherited disorders. However, this is a category of disease for which there is at present only the nonspecific and partially successful treatment of corneal transplantation. Retroviral

vectors are likely to ultimately enable this approach to be feasible. The cornea remains an ideal model tissue for genetic modification with great potential to assume a vanguard role in the

development of successful new gene therapies. Much progress has been made in the genetic modification of the epithelium, endothelium, and keratocytes using viral and nonviral vectors in a

variety of model systems. Despite these advances, continued improvements in existing or new gene therapy techniques will be required before these approaches can be applied to human clinical

trials. REFERENCES * Fabre JW . Prospects for genetic manipulation of donor organs and tissues. _Transplantation_ 2001; 71: 1207–1209. Article  CAS  PubMed  Google Scholar  * Bron AJ .

Genetics of the corneal dystrophies: what we have learned in the past twenty-five years? _Cornea_ 2000; 19: 699–711. Article  CAS  PubMed  Google Scholar  * Biswas S, Munier F, Yardley J,

Hart-Holden N, Perveen R, Cousin P _et al_. Missense mutations in _COL8A2_, the gene encoding the α2 chain of type VIII collagen, cause two forms of corneal endothelial dystrophy. _Hum Mol

Genet_ 2001; 10: 2415–2423. Article  CAS  PubMed  Google Scholar  * Bertelmann E, Jaroszewski J, Pleyer U . Corneal allograft rejection: current understanding. _Ophthalmologica_ 2002; 216:

2–12. Article  PubMed  Google Scholar  * Carr DJJ, Härle P, Gebhardt BM . The immune response to ocular herpes simplex virus type 1 infection. _Exp Biol Med_ 2001; 226: 353–366. Article  CAS

  Google Scholar  * Dunaief JL, Ng EW, Goldberg MF . Corneal dystrophies of epithelial genesis: the possible therapeutic use of limbal stem cell transplantion. _Arch Ophthalmol_ 2001; 119:

120–122. CAS  PubMed  Google Scholar  * Tanelian DL, Barry MA, Johnston SA, Le T, Smith G . Controlled gene gun delivery and expression of DNA within the cornea. _Biotechniques_ 1997; 23:

484–488. Article  CAS  PubMed  Google Scholar  * Tsubota K, Inoue H, Ando K, Ono M, Yoshino K, Saito I . Adenovirus-mediated gene transfer to the ocular surface epithelium. _Exp Eye Res_

1998; 67: 531–538. Article  CAS  PubMed  Google Scholar  * Larkin DFP, Oral HB, Ring CJA, Lemoine NR, George AJT . Adenovirus-mediated gene delivery to the corneal endothelium.

_Transplantation_ 1996; 61: 363–370. Article  CAS  PubMed  Google Scholar  * Stechschulte SU, Joussen AM, von Recum HA, Poulaki V, Moromizato Y, Yuan J _et al_. Rapid ocular angiogenic

control via naked DNA delivery to the cornea. _Invest Ophthalmol Vis Sci_ 2001; 42: 1975–1979. CAS  PubMed  Google Scholar  * Seitz B, Moreira L, Baktanian E, Sanchez D, Gray B, Gordon EM

_et al_. Retroviral vector-mediated gene transfer into keratocytes _in vitro_ and _in vivo_. _Am J Ophthalmol_ 1998; 126: 630–639. Article  CAS  PubMed  Google Scholar  * Kampmeier J,

Behrens A, Wang Y, Yee A, Anderson WF, Hall FL _et al_. Inhibition of rabbit keratocyte and human fetal lens epithelial cell proliferation by retrovirus-mediated transfer of antisense cyclin

G1 and antisense MAT1 constructs. _Hum Gene Ther_ 2000; 11: 1–8. Article  CAS  PubMed  Google Scholar  * Behrens A, Gordon EM, Li L, Liu PX, Chen Z, Peng H _et al_. Retroviral gene therapy

vectors for prevention of excimer laser-induced corneal haze. _Invest Ophthalmol Vis Sci_ 2002; 43: 968–977. PubMed  Google Scholar  * Kamata Y, Okuyama T, Kosuga M, Ohira A, Kanaji A,

Sasaki K _et al_. Adenovirus-mediated gene therapy for corneal clouding in mice with mucopolysaccharidosis type VII. _Mol Ther_ 2001; 4: 307–312. Article  CAS  PubMed  Google Scholar  * Wang

X, Appukuttan B, Ott S, Patel R, Irvine J, Song J _et al_. Efficient and sustained transgene expression in human corneal cells mediated by a lentiviral vector. _Gene Ther_ 2000; 7: 196–200.

Article  CAS  PubMed  Google Scholar  * Fehervari Z, Rayner SA, Oral HB, George AJT, Larkin DFP . Gene transfer to _ex vivo_ stored corneas. _Cornea_ 1997; 16: 459–464. Article  CAS  PubMed

  Google Scholar  * Oral HB, Larkin DFP, Fehervari Z, Byrnes AP, Rankin AM, Haskard DO _et al_. _Ex vivo_ adenovirus-mediated gene transfer and immunomodulatory protein production in human

cornea. _Gene Ther_ 1997; 4: 639–647. Article  CAS  PubMed  Google Scholar  * Tsai ML, Chen SL, Chou PI, Wen LY, Tsai RJF, Tsao YP . Inducible adeno-associated virus vector-delivered

transgene expression in corneal endothelium. _Invest Ophthalmol Vis Sci_ 2002; 43: 751–757. PubMed  Google Scholar  * Bainbridge JWB, Stephens C, Parsley K, Demaison C, Halfyard A, Thrasher

AJ _et al_. _In vivo_ gene transfer to the mouse eye using an HIV-based lentiviral vector; efficient long-term transduction of corneal endothelium and retinal pigment epithelium. _Gene Ther_

2001; 8: 1665–1668. Article  CAS  PubMed  Google Scholar  * Pleyer U, Groth D, Hinz B, Keil O, Bertelmannn E, Rieck P _et al_. Efficiency and toxicity of liposome-mediated gene transfer to

corneal endothelial cells. _Exp Eye Res_ 2001; 73: 1–7. Article  CAS  PubMed  Google Scholar  * Hudde T, Rayner SA, Comer RM, Weber M, Isaacs JD, Waldmann H _et al_. Activated polyamidoamine

dendrimers, a non-viral vector for gene transfer to the corneal endothelium. _Gene Ther_ 1999; 6: 939–943. Article  CAS  PubMed  Google Scholar  * Klebe S, Sykes PJ, Coster DJ, Bloom DC,

Williams KA . Gene transfer to ovine corneal endothelium. _Clin Exp Ophthalmol_ 2001; 29: 316–322. Article  CAS  PubMed  Google Scholar  * Hart SL, Arancibia-Cárcamo CV, Wolfert MA, Mailhos

C, O'Reilly NJ, Ali RR _et al_. Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector. _Hum Gene Ther_ 1998; 9: 575–585. Article  CAS  PubMed  Google

Scholar  * Tan PH, King WJ, Chen D, Awad HA, Mackett M, Lechler RI _et al_. Transferrin receptor-mediated gene transfer to the corneal endothelium. _Transplantation_ 2001; 71: 552–560.

Article  CAS  PubMed  Google Scholar  * Larkin DF, Calder VL, Lightman SL . Identification and characterization of cells infiltrating the graft and aqueous humour in rat corneal allograft

rejection. _Clin Exp Immunol_ 1997; 107: 381–391. Article  CAS  PubMed  PubMed Central  Google Scholar  * Comer RM, King WJ, Ardjomand N, Theoharis S, George AJT, Larkin DFP . Effect of

administration of CTLA4-Ig as protein or cDNA on corneal allograft survival. _Invest Ophthalmol Vis Sci_ 2002; 43: 1095–1103. PubMed  Google Scholar  * Rayner SA, King WJ, Comer RM, Larkin

DFP, George AJT . Pulsatile local bioactive tumor necrosis factor in allograft rejection. _Clin Exp Immunol_ 2000; 122: 109–116. Article  CAS  PubMed  PubMed Central  Google Scholar  *

Rayner SA, Larkin DFP, George AJT . TNF receptor secretion after _ex vivo_ adenoviral gene transfer to cornea and effect on _in vivo_ graft survival. _Invest Ophthalmol Vis Sci_ 2001; 42:

1568–1573. CAS  PubMed  Google Scholar  * Arancibia-Cárcamo CV, Oral HB, Haskard DO, Larkin DFP, George AJT . Lipoadenofection-mediated gene delivery to the corneal endothelium: prospects

for modulating graft rejection. _Transplantation_ 1998; 65: 62–67. Article  PubMed  Google Scholar  * Klebe S, Sykes PJ, Coster DJ, Krishnan R, Williams KA . Prolongation of sheep corneal

allograft survival by _ex vivo_ transfer of the gene encoding interleukin-10. _Transplantation_ 2001; 71: 1214–1220. Article  CAS  PubMed  Google Scholar  * Noisakran S, Carr DJJ . Topical

application of the cornea post-infection with plasmid DNA encoding interferon-α1 but not recombinant interferon-_α_A reduces herpes simplex virus type 1-induced mortality in mice. _J

Neuroimmunol_ 2001; 121: 49–58. Article  CAS  PubMed  Google Scholar  * Inoue T, Inoue Y, Nakamura T, Yoshida A, Takahashi K, Inoue Y . Preventive effect of local plasmid DNA vaccine

encoding gD (HSV-1) or gD-IL-2 on herpetic keratitis. _Invest Ophthalmol Vis Sci_ 2000; 41: 4209–4215. CAS  PubMed  Google Scholar  * Inoue T, Inoue Y, Hayashi K, Yoshida A, Nishida K,

Shimomura Y _et al_. Topical administration of HSV gD-IL-2 DNA is highly protective against murine herpetic stromal keratits. _Cornea_ 2002; 21: 106–110. Article  PubMed  Google Scholar 

Download references ACKNOWLEDGEMENTS Supported by The Special Trustees of Moorfields Eye Hospital (Grant LARF1010, DFPL), the Mr. and Mrs. Raymond P.L. Kwok Research Fund (ASJ), and the

Niarchos Foundation (ASJ). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Cornea and External Disease Service, Moorfields Eye Hospital, London, UK A S Jun & D F P Larkin * Cornea and

External Disease Service, The Wilmer Eye Institute, The Johns Hopkins Medical Institutions, Baltimore, 21287, MD, USA A S Jun Authors * A S Jun View author publications You can also search

for this author inPubMed Google Scholar * D F P Larkin View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to D F P

Larkin. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Jun, A., Larkin, D. Prospects for gene therapy in corneal disease. _Eye_ 17, 906–911 (2003).

https://doi.org/10.1038/sj.eye.6700565 Download citation * Received: 28 February 2003 * Accepted: 28 February 2003 * Published: 20 November 2003 * Issue Date: 01 November 2003 * DOI:

https://doi.org/10.1038/sj.eye.6700565 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not

currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * cornea * epithelium * endothelium * keratocyte * gene

therapy * vectors