AU2006202007A1 - Tissue modification - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT

(ORIGINAL)

IND

Name of Applicants: Actual Inventors: Douglas John Coster of 3 Newcastle Street, Heathpool, South Australia 5068, Australia AND Keryn Anne Williams of 17 East Parade, Kingswood, South Australia 5062, Australia AND Sonja Klebe of 4 Yatina Road, Aldgate, South Australia 5154, Australia Douglas John Coster Keryn Anne Williams Sonja Klebe Address for Service: DAVIES COLLISON CAVE, Patent Trademark Attorneys, of 1 Nicholson Street, Melbourne, 3000, Victoria, Australia Ph: 03 9254 2777 Fax: 03 9254 2770 Attorney Code: DM Invention Title: "Tissue modification" The following statement is a full description of this invention, including the best method of performing it known to us:- -1- TISSUE MODIFICATION This is a divisional of Australian patent application No. 79432/01, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION The present invention relates generally to the field of tissue modification. More particularly, the present invention contemplates methods of modifying cells of corneal tissue to express an active agent. The present invention further provides modified corneal tissue and vectors utilised in such methods and provides methods of xeno- and allotransplantation utilising the modified corneal tissue.

BACKGROUND OF THE INVENTION There are numerous diseases and disorders that can effect corneal tissue and which can, as a result, adversely effect or eliminate vision. For example, allergies, conjunctivitis, corneal infections, Fuchs' dystrophy (deterioration of corneal endothelial cells), varicella-zoster virus, iridocorneal endothelial syndrome, keratoconus, ocular herpes and a number of other conditions, as well as congenital corneal abnormalities can be responsible for corneal damage or irregularity that may affect vision. In an endeavour to restore sight or improve vision in people suffering from corneal abnormalities it has become particularly common to perform corneal transplant operations where the abnormal corneal tissue is removed and replaced using fine sutures with normal corneal tissue obtained from a donor. Although corneal transplant operations enjoy a high rate of success there are nonetheless some p'roblems which can occur, such as rejection of the replacement comea and ocular fibrosis or scaring. Even in the case of a successful corneal transplantation it is necessary for subsequent administration of immnunomodulatory agents. Non-compliance by the patient with prescribed dosing regime of immunomodulating agents may give rise to tissue rejection. There is, accordingly, a need for improved means of prolonging corneal graft survival and preventing tissue rejection as well as for the provision of approaches for therapy of ocular infection, wounds and fibrosis and for therapy of other ocular disorders, for example.

C \TEMP 2o.l Ip2 doc.-I IlllA)i -2- The cornea is a highly organised group of cells and proteins which unlike most tissue is clear, and does not contain blood vessels to nourish or protect against infection. The cornea receives nutritional supply from tears and the aqueous humor found in the anterior chamber located behind it. The cornea is composed of five basic layers, namely the protective external epithelium, Bowman's layer which is located below the epithelial basement membrane and is composed of collagen fibers, the stroma which comprises about of the cornea's thickness and consists primarily of water and collagen Descemet's membrane located beneath the stroma, which is composed of collagen fibers produced by the endothelial cells located in the inner most layer of the cornea, the lower endothelium.

The endothelial cells are essential in maintaining clarity of the cornea by removing excess fluid from the stroma. Endothelial cells are non-regenerative and once damaged, corneal transplantation is the only means of replacement.

Irreversible immunological rejection is the major cause of human corneal graft failure despite the immunologically privileged nature of the eye The histological correlates of rejection include local upregulation of major histocompatability complex and adhesion molecules, an influx of mononuclear cells into the cornea and anterior chamber, and local production of some inflammatory cytokines The major target of corneal graft rejection is the corneal endothelium. Human (but not rodent) corneal endothelium is essentially amitotic so that damage to the monolayer during graft rejection cannot be repaired.

Gene therapy has the potential to influence an allograft response through local expression of a modulatory gene product within transplanted donor tissue. The present inventors consider that the cornea may be uniquely amenable to such an approach because of its small size, which may allow modification of the whole tissue, and because of the ease with which a donor cornea may be manipulated in vitro and stored for a considerable period (for example, up to 28 days) prior to transplantation. The anatomical location and clarity of the cornea allow in vivo assessment of the entire graft in the post-operative period and loss of function is easy to detect. Furthermore, the cornea and anterior chamber are at least partially immunologically privileged sites which may allow the use of otherwise C \TEMP\cornc.il p2 doc- 1llln/l N. -3immunogenic or pro-inflammatory vectors.

Both cellular (44-46) and viral IL-10 (43, 47-49) have been reported to prolong allograft survival and to modulate chronic rejection in a variety of small animal models, and gene knock-out mice show decreased cardiac allograft survival and increased evidence of chronic rejection (50, 51). However, in at least one report, systemic administration of murine IL-10 was shown to exacerbate murine cardiac allograft rejection and further, subconjunctival and systemic administration of various doses of murine IL-10 has been shown to be ineffective in prolonging corneal graft survival in the rat The effect of IL-10 on allograft survival appears to be dependent upon both timing of administration and upon dose Mouse and human IL-10 may not be entirely homologous with respect to function: in particular, the former can be immunostimulatory for murine T cells a property not shared by human ILl-10 or viral IL-10, although it has been suggested informally that endotoxin levels in various cytokine preparations may Jave affected some results.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

SUMMARY OF THE INVENTION Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

According to one embodiment of the present invention there is provided a method of modifying cells of corneal tissue such that said cells produce an active agent, said method comprising introducing into said cells an effective amount of an expression vector which comprises a nucleotide sequence encoding said active agent and culturing said cells for a C \TEMcmzorI p 2 tm. Idoci IAI Q-4time and under conditions sufficient for said cells of said corneal tissue to express said N nucleotide sequence and produce said active agent such that cells of said corneal tissue will express the active agent and prolong corneal graft survival, improve corneal graft healing or prevent corneal tissue rejection as well as for the provision of therapy for ocular infection, wounds, fibrosis or other ocular disorders.

IDAccording to another embodiment of the present invention there is provided a corneal tissue comprising cells modified to express a nucleotide sequence to produce an active agent, said nucleotide sequence is not expressed in normal corneal tissue or is expressed at elevated levels relative to normal corneal tissue.

In another embodiment of the invention there is provided a corneal tissue comprising cells modified to produce an active agent, wherein modification is by exposing harvested corneal tissue to an effective concentration for transfection of an expression vector which comprises a nucleotide sequence encoding for the active agent, for a period and under conditions sufficient to allow transfection.

In a further embodiment of the invention there is provided a method of improving corneal graft healing and/or prolonging graft survival comprising modifying cells of corneal tissue such that said cells produce an active agent, said method comprising introducing into said cells an effective amourlt of an expression vector which comprises a nucleotide sequence encoding said active agent and culturing said cells for a time and under conditions sufficient for said cells of said corneal tissue to express said nucleotide sequence and produce said active agent, and then transplanting the corneal tissue to an eye of a recipient.

In a still further embodiment of the invention there is provided an expression vector for use in modifying corneal tissue to express a nucleotide sequence to produce an active agent, said nucleotide sequence is not expressed in normal corneal tissue or is expressed at elevated levels relative to normal corneal tissue Preferably the active agent is a peptide hormone, a cytokine or an analogue thereof. In a C \TEMPcomnaj tp2 doc-I l/I0/II preferred embodiment of the invention the cytokine is an interleukin, an interferon or a growth factor, or an analogue thereof. In preferred embodiments of the invention the cytokine is selected from IL-10, IL-4, the P-40 component of IL-12, Bcl2, interferon y, interferon a and TGF p.

In preferred embodiments of the invention the corneal tissue is harvested from a mammal, particularly preferably from a human. Preferably the recipient of transplanted corneal tissue is a mammal, particularly preferably a human. In a particularly preferred embodiment of the invention corneal tissue is harvested from a human and transplanted to another human recipient.

In a preferred embodiment of the invention the corneal tissue cells modified are epithelial cells, stroma cells and/or endothelial cells. Particularly preferably the modified cells are endothelial cells. In a preferred embodiment of the invention preferably at least more preferably at least 20%, particularly preferably at least 30% or at least 50% and most particularly preferably at least 70% of corneal endothelial cells are modified by methods according to the invention.

In preferred embodiments of the invention the expression vector is a viral, bacterial or plasmid vector. In particularly preferred embodiments of the invention the expression vector is an adeno-associated viral vector or an adenoviral vector.

Preferably the effective concentration for infection is between about 1 x 105 to 1 x particle forming units (pfu) per cornea. Particularly preferably the effective concentration for infection is between about 5 x 105 to 5 x 108 pfu/cornea, more particularly preferably between about 2 x 106 and about 9 x 10 7 pfu/corea.

In a preferred embodiment of the invention the period sufficient to allow infection is between about 1 minute and about 48 hours, particularly preferably between about minutes and 24 hours, more particularly preferably between about 30 minutes and 6 hours and most particularly preferably between about 1 hour and about 3 hours.

C \TEM~ ornel p2 doc-l -6- In another preferred embodiment of the invention the expression vector comprises nucleotide sequences encoding for two or more active agents.

BRIEF DESCRIPTION OF THE FIGURES The invention will be further described, by way of example, with reference to the following figures: Figure 1 is a photographic representation of autoradiographs showing uptake of 3

H

thymidine in organ-cultured ovine corneal endothelium, 3 days after deliberate injury.

FIGURE 1(A) shows uptake in endothelial cells (arrowed) at margins of deliberate injury; magnification x32; FIGURE 1(B) shows high power view showing mitotic figure (arrowed); magnification x128.

Figure 2 is a graphical representation showing the effect of viral concentration and incubation time with virus on transfection efficiency of the adenoviral vector Ad-lacZ for ovine comeal endothelium, and stability of expression of p-galactosidase in endothelial cells of organ-cultured ovine corneas. In each instance, reporter gene expression was quantified by counting P-galactosidase-positive cells in three representative areas per cornea. FIGURE 2(A) shows concentrations of Ad-lacZ from 6.6 x 102 to 6.6 x 108 pfu per cornea were used to transfect ovine corneas in vitro under otherwise identical conditions.

Corneas were harvested 48 hours later. Each bar represents the mean percentage positive cells SD of counts from 3 to 6 corneas. FIGURE 2(B) shows ovine corneas were incubated with Ad-lacZ at 6.6 x 10 6 and 6.6 x 10 7 pfu per cornea for 0.5-2.0 h. Corneas were harvested 48 hours later. Each bar represents the mean percentage of positive cells SD from 3 organ-cultured corneas. FIGURE 2(C) shows duration of expression of 3galactosidase in organ-cultured ovine corneas after transfection with 6.6 x 106 Ad-lacz pfu per cornea for 2 hours. Corneas were harvested at the indicated time-points. Each point represents the mean percentage positive cells SD of 3-14 corneas.

C \TEMPForneal Ip2 doc- ll/11ill -7- Figure 3 is a photographic representation of agarose 1.5% w/v gel showing product for ILand GAPDH in sheep corneas transfected with Ad-IL-10 under optimal conditions and organ-cultured for 21 days prior to RNA extraction and RT-PCR. Dilutions of cDNA at 1/1, 1/10 and 1/100 dilutions were run in duplicate lanes. Lanes marked "no DNA" represent controls in which water replaced cDNA.

Figure 4 is a representation showing outcome of gene-modified penetrating corneal allografts in sheep: FIGURE 4(A) shows rejecting unmodified allograft, day 29 post-graft; FIGURE 4(B) shows surviving IL-10-modified allograft, day 190 post-graft.

DETAILED DESCRIPTION OF THE INVENTION The present invention is predicated in part on the development of an approach for treating ocular disorders in animals and humans. In the specifically exemplified embodiments of the present invention harvested corneal tissue has been modified to express immunomodulatory agents having the effect of prolonging corneal graft survival once implanted into a recipient, relative to the expected survival time in the case where no immunomodulatory agent is administered. However, the present invention extends to its use in association with corneal allo- or xeno-transplantation techniques. Methods according to the present invention may be adopted for treatment of other ocular disorders such as the treatment of ocular wounds, infections or fibrosis or of other ocular diseases such as glaucoma, keratoconus, corneal dystrophies, corneal infections, tumours of the eye, proliferative lesions, pterygium and inflammatory disorders of the eye including Stevens- Johnson syndrome and mucous panphigoid. Reference to "treatment" is intended to include both therapeutic and prophylactic treatments.

One aspect of the invention relates to methods of modifying cells of corneal tissue to produce an active agent. As the nucleotide sequence for these active agents are to be expressed in the corneal tissue cells as a result of gene transfer, the active agents will of C VTEMPcorncaltp2 doc-. 11I/Ill -8course constitute peptides, polypeptides or proteins, the expression of which can be Sencoded for by nucleotide sequences, particularly DNA sequences. The term "expression" includes translation into an expression product, generally a protein. However, the present invention extends to expression products in the form of spliced out introns which might be S 5 involved in regulatory mechanisms within the cell. Collectively, the active agents, regardless of the amino acid sequence, may be referred to herein as "peptides". The active \agents according to the invention may for example constitute naturally occurring or synthetic peptide hormones, cytokines or analogues thereof. By use of the term "analogue" it is intended to embrace modified forms of naturally occurring or synthetic peptide hormones or cytokines having physiological activity which may for example be modified relative to the molecule upon which they are based by the addition, deletion or substitution of single or multiple amino acids.

Examples of active agents that may be produced by the methods and vectors according to the present invention include peptide hormones and cytokines and analogues thereof which may for example have immunomodulatory, anti infective, tissue regeneration, wound healing or fibrosis reduction activity. Cytokines that may be adopted in the present invention include those selected from the interleukins, the interferons and the growth factors as well as analogues thereof. Specific examples of cytokines that may be adopted include IL-10, IL-4, the P-40 component of IL-12, Bcl2, interferon y, interferon a and TGFp. Without wishing'to limit the invention to any one theory or mode of action, the Pcomponent of IL-12 is believed to act as an IL-12 Receptor antagonist, thus blocking the effects of IL-12. Whereas IL-10 is believed to act as an agonist to reduce or inhibit macropahge activation and inflammation. Bcl2 is an example of an anti-apoptotic agent, which may act by protecting against growth factor withdrawal. It is to be understood however that these specific cytokines are mentioned as active agents by way of example only, and that other peptide agents with useful activity may equally be adopted. Examples of such agents are low MW aqueous humor peptide anti-inflammatory agents, antiapoptotic agents, agents capable of down regulating class I or class II MHC antigens, agents capable of inhibiting NK cells or Macrophage or other agents involved in immune C:\TEMP.orr- p2 dx- I111A) I -9privilege or active down regulation or prevention of the DTH response, such as are produced by suppressor T cells.

The corneal tissue to be modified according to the present invention will generally have been harvested from a donor, usually a donor mammal. Preferably the donor will be selected from the same species as the corneal transplant recipient and generally from a donor having matching tissue and/or blood types as the intended recipient, as well understood in the art. There may, however, be circumstances, such as if there is insufficient donor organ supply from members of the same species (allo-transplantation), where corneal tissue is harvested from a donor member of another species (xenotransplantation). In the case of xeno-transplantation the tissue donor may be an animal that has been genetically modified to remove or reduce the impact of species specific immunogenic differences. The present invention extends to modifying corneal tissue to enable production by organ culture techniques which should then similarly be harvested for modification by methods according to the present invention. Also artificial corneas comprising cells should similarly be modified according to the present invention.

Mammals from which corneal tissue may be harvested and/or to which corneal tissue may be transplanted include, but are not limited to, humans, farm animals including cattle, sheep, goats, pigs, horses, etc.; captive wild animals including lions, tigers, deer, chimpanzees, apes, gorillas, baboons, etc.; domestic animals such as cats and dogs, etc, or laboratory animals such as rabbits, mice, guinea pigs, rats and the like. Preferably the c6drneal tissue is harvested from a human donor for transplant to a human recipient. In the case of human corneal tissue donors, the donor will generally be a person registered as an organ donor who has met an untimely death, and whose corneal tissue is in good condition.

In the case of animal donors, the animal may be sacrificed in order to harvest the corneal tissue or may in fact be sacrificed for other purposes such that the corneal tissue becomes available.

The corneal tissue will preferably be obtained from the donor within a relatively short period post mortem, preferably within three to four hours and particularly preferably C \TEMPcorrn Ip 2 d.-I IIPH( within the first hour. The conditions under which the corneal tissue should be removed from the donor and maintained prior to modification are a matter of routine and are well understood by persons skilled in the art. Naturally, the use of a suitable tissue culture media is required to maintain the tissue in a healthy state prior to modification and transplantation. Preferably modification of the corneal tissue will be conducted within a matter of a few hours from harvesting of the tissue, although it is possible to maintain corneal tissue under tissue culture conditions for up to about 28 days.

The expression vector may be of any type and include viral, bacterial and plasmid expressing vector systems. Furthermore, naked nucleic acid molecules may be used which may integrate into the genome or which are transiently expressed. Examples of suitable viral vectors include HSV, lentivirus, retroviral vectors, fowlpox vectors and adeno associated viral vectors. Preferred vectors are adenoviral vectors. Naturally, the expression vector adopted result in expression in corneal tissue cells and particularly cells of the corneal epithelium, stroma and/or endothelium. Preferably expression of the active agent, through infection by the expression vector, is within the endothelial cells and particularly preferably the level of infection of these cells with the selected expression vector is such that active ingredient expression is demonstrated in at least preferably at least more preferably at least 20%, particularly preferably at least 30% or at least 50% and most particularly preferably at least 70% of corneal endothelial cells. The expression vector selected will of course include all of the features required for expression of protein in a mammalian cell. For example, preferred expression vectors will contain a molecular chimera containing the coding sequence of active agent or agents selected, an appropriate polyadenylation signal for a mammalian gene a polyadenylation signal which will function in a mammalian gene), and suitable enhancers and promoter sequences in the correct orientation.

In mammalian cells, normally two DNA sequences are required for the complete and efficient transcriptional regulation of genes that encode messenger RNAs in mammalian cells: promoters and enhancers. Promoters are located immediately upstream from the start side of transcription. Promoter sequences are required for accurate and efficient C \TEMP.orel Ip2 doc-.i I(Lmi -Ilinitiation of transcription. Different gene-specific promoters reveal a common pattern or organisation. A typical promoter includes an AT-rich region called a TATA box (which is located approximately 30 base pairs 5' to the transcription initiation start site) and one or more upstream promoter elements (UPE). The UPEs are a principle target for the interaction with sequence-specific nuclear transcription factors. The activity of promoter sequences is modulated by other sequences called enhancers. The enhancer sequence may be a great distance from the promoter in either upstream or downstream position.

Hence, enhancers operate in an orientation- and position-independent manner. However, based on similar structural organisation and function that may be interchanged the absolute distinction between promoters and enhancers is somewhat arbitrary. Enhancers increase the rate of transcription from the promoter sequence. The necessary machinery required for cellular expression of the active agent or agents must of course be located in the appropriate orientation with regard to the nucleotide sequence (preferably DNA) that encodes the active agent or agents that has been inserted into the expesion vector by the use of routine molecular biology techniques, such as for example as further explained in Ausubel et al (1987) in: Current Protocols in Molecular Biology, Wyle Interscience (ISBN 047150338) the disclosure which is included herein in its entirety by way of reference.

Also mentioned by way of reference in relation to preparation of expression vectors, the disclosure of which is included herein by reference is He et al, "A simplified system for generating recombinant adenovirus", Proc. Nat. Acad Sci 1998, 95: 2509-2514. The expression vector may appropriately include a suitable nuclear localisation signal and will be propagated in any permissive cell line. Permissive cell lines, mentioned by way of example only, include EIA and E1B trans-complementing 293 cells. Other cell lines will equally be useful for propagation of expression vectors according to the invention, as would clearly be understood by persons skilled in the art.

The exposure of corneal tissue to expression vectors according to the invention will be in a manner that will allow infection by the expression vector of the corneal tissue cells. The exposure of the corneal tissue to the expression vector may simply be by including the expression vector into the corneal tissue culture media. Other means of exposure such as via direct injection of the expression vectors into the corneal tissue or via high velocity C \TEMP',orc.pl tp dc-I II/tl.

-12bombardment may also be adopted, although care should be taken to avoid damage to the corneal tissue. Liposomal agents may also be used to transfer genes into the epithelium (14, 15). To ensure adequate levels of infection of corneal cells with the expression vector it is necessary for an effective concentration for infection of the expression vector to be utilised. For example, concentrations of between about 1 x 105 and about 1 x 1010 particle forming units (pfu) per cornea may be adopted. Preferably the effective concentration is between about 5 x 10 5 and 5 x 108 pfu/cornea, particularly preferably between about 2 x 106 and about 9 x 107 pfu per cornea. It is also important that the exposure of the corneal tissue to the expression vector is for a period sufficient to allow infection, such as for example between about 1 minute and about 48 hours, preferably between about 10 minutes and 24 hours, more preferably between about 30 minutes and about 6 hours and most preferably between about 1 hour and about 3 hours. This can, for example, be achieved by simply introducing the expression vector into the corneal tissue culture media and then changing the media to remove non-infected vector after the appropriate period, optionally with one or more washing stages.

The active agent which the corneal cells are modified to express may be one which is not expressed by normal corneal tissue or which, following modification is expressed at elevated levels relative to those of normal corneal tissue.

Also encompassed within the scope of the present invention are processes of improving corneal graft healing and/or prolonging graft survival involving the use of corneal tissue modified according to methods discussed above. When referring to "improving corneal graft healing" and "prolonging graft survival" these terms are intended to be relative to the rate and extent of healing and the duration of graft survival expected by conducting corneal allo- or xeno- transplantation, without the administration to the patient of other graft healing or immunomodulatory agents.

Also included within the scope of the invention are the expression vectors prepared for use in methods according to the invention which allow for expression of active agents within C \TEIMPt, orn l p2 doc-I 1/lItll 13 corneal tissue cells, as well as the corneal tissue which comprises cells modified to express active agents.

The present invention will now be described further, by way of example only with reference to the following examples:

EXAMPLES

Example 1 The inventors selected a model of orthotopic corneal transplantation in the outbred sheep, a relevant preclinical model in which unmodified comeal allografts undergo rejection at three weeks post-operatively in a manner that is very similar at a clinical level to human corneal graft rejection Adenoviral vectors have already been shown to be capable of transferring reporter genes into corneal endothelium of various species (10-13) and the use of liposomal agents has also previously been explored (14, 15). Given that the mitotic potential of sheep corneal endothelium was unknown, the replicative capacity of this tissue was first examined, to allow an informed choice of the vector for gene therapy to be made.

Materials and Methods Ovine corneal organ-culture. Fresh sheep eyes obtained within 3 hr of donor death from a local abattoir (Lobethal Abattoirs, Lobethal, SA, Australia) were decontaminated for 3 min in 10% w/v povidone-iodine (Faulding Pharmaceuticals, Salisbury, SA, Australia) and ufiderwent two washes by immersion in sterile 0.9 w/v NaCl. A limbal incision was made with a scalpel blade and the cornea with a 2 mm scleral rim was removed with corneal scissors. Corneas were organ-cultured in 15 ml complete medium (HEPESbuffered RPMI medium (ICN, Costa Mesa, CA, USA) supplemented with 10% v/v heatinactivated (56 0 C, 30 min) fetal calf serum (FCS), 100 IU/ml penicillin, 100 i.g/ml streptomycin, 2.5 pg/ml amphotericin B and 2 mM L-glutamine (all from Gibco BRL, Gaithersburg, MD, USA)) at 32 0 C in air for up to 28 days. Medium was changed twice weekly.

C \TEMpcornl p2 doc-I I1/IOm) -14- Evaluation of the mitotic potential of ovine corneal endothelium. A 4 mm long central cross-shaped defect was produced with a 27 gauge needle on endothelial monolayers of fresh ovine corneas. Corneas were then placed in sterile shallow wells, endothelium facing upward. 500 l1 complete medium containing 25 pCi 6-3H thymidine (TRA61; Amersham, Little Chalfont, Buckinghamshire, UK) was placed in the corneal cup for 5 hr at 32 0

C.

The solution was then diluted to a total volume of 3 ml with complete medium containing no isotope and to 10 ml total after 24 hr. After 3 days, coreas were harvested, fixed in 3:1 absolute ethanol:glacial acetic acid at room temperature for 24 hr and transferred to ethanol for a further 24 hr. Corneal endothelium was removed by blunt dissection through the stroma, mounted on gelatin-coated slides and air-dried for 2 hr. Flat-mounts were coated with LM-1 photographic autoradiography emulsion (Amersham, Little Chalfont, Buckinghamshire, UK), exposed at 4 0 C for 4 weeks and processed according to the manufacturer's protocol. The flat-mounts were stained with Giemsa and mounted in Depex (BDH Chemicals, Kilsyth, VIC, Australia). As negative controls, corneas were injured and incubated in 3 H thymidine-free medium, and uninjured corneas were incubated with and without 3 H thymidine. Corneal epithelial flat-mounts prepared from corneas incubated as above with the epithelial surface in contact with tritiated thymidinecontaining medium were used as a positive control.

Replicative capacity of ovine corneal endothelium. Ovine corneal endothelium was deliberately injured and the corneas organ-cultured in the presence of 3 H thymidine. The site of injury was still clearly visible at the light microscope on corneal endothelial flatmounts harvested after 3 days in organ-culture. Uptake of H thymidine into the nuclei of the occasional endothelial cell close to the site of the injury was observed (Fig. 1A) and very rare mitotic figures were identified (Fig. 1B). Uptake of 3 H thymidine was limited to the close vicinity of the injury: no uptake occurred in the corneal periphery. Uninjured comeas (negative control) showed no uptake of 3 H thymidine and corneas incubated with the epithelial surface in contact with isotope-containing solution (positive control) showed substantial uptake (not shown). The data suggested that the replicative capacity of ovine comeal endothelium was very limited and that, for example, replication-deficient C \TEMPoincal Ip2 doc.l 1/1II1/ adenoviral virus (which remains episomal) would thus be a suitable vector for gene transfer to ovine endothelium.

3H thymidine is incorporated into DNA by proliferating cells and can be visualised by autoradiography. This method has been used previously (30-33) to investigate the mitotic activity of corneal endothelium in many species. In the sheep cornea, uninjured endothelia incubated with 3 H thymidine did not take up the isotope. Localized uptake into single endothelial cells was observed after deliberate injury, but proliferation was insufficient to cover the defect over three days. Our data suggest that ovine corneal endothelium has a very limited mitotic potential: some proliferation can be induced by a triggering event, but does not otherwise occur. For practical purposes, then, ovine corneal endothelium may be considered essentially amitotic.

Transfection of ovine corneal endothelium with adenoviral vectors, The replication deficient El-, E3-deleted adenovirus type 5 vectors encoding E. coli lacZ under the transcriptional control of the CMV promoter (Ad-lacZ), or containing an empty plasmid (Ad-mock), or encoding full-length ovine IL-10 (Ad-IL-10) or P-40 subunit of IL-12 (Ad- P40-IL-12) where prepared following the approach as described in Hu et al. as referenced above. cDNA sequences for these species are available on public databases. The Ad-lacZ construct contained a nuclear localization signal. Vectors were propagated in EIA, EIB trans-complementing 293 cells following standard protocols (18-20). In order to determine optimal viral concentration for infection of corneal endothelial cells, corneas were infected with concentrations of Ad-lacZ ranging from 6.6 x 102 6.6 x 108 plaque forming units (pfu) per cornea in complete medium. Control corneas were uninfected or similarly infected with Ad-mock. Optimal infection time was determined by incubation of the corneas with 6.6 x 10 6 and 6.6 x 10 7 pfu Ad-lacZ per cornea for 0.5, 1, 1.5 and 2 hours; the vector was then diluted out and the corneas were re-incubated for a further 48 hr in 15 ml complete medium. To examine duration of reporter gene expression, corneas infected with 6.6 x 10 pfu per cornea for 2 hours at room temperature were organ-cultured for 2 days (n 14), 3 days (n 6 days (n 7 days 10 days (n 13 days (n 14 C \TEM Pornal ip2 doc-l IlI)o I -16days (n 16 days (n 21 days (n and 28 days (n After incubation, all corneas were processed for lacZ reporter gene expression.

Detection of lacZ reporter gene expression. Prior to processing, corneas were fixed in 2.5% formaldehyde and 0.25% glutaraldehyde in Dulbecco's A phosphate-buffered saline (PBS) for 15 min on ice followed by two 15 min washes in PBS on ice to inactivate the viral vector and inhibit endogenous 3-galactosidase Expression of E. coli pgalactosidase was detected using 2.5 ml/cornea of a solution of I mg/ml 5-bromo-4-chloro- 3-indoxyl-P-D-galactoside (ICN, Costa Mesa, CA, USA), N-dimethylformamide (BDH Chemicals, Kilsyth, VIC, Australia), 2 mM MgCI 2 5 mM K 4 Fe(CN) 6 5 mM K 3 Fe(CN) 6 in PBS-2 (16 mM Na 2

HPO

4 4 mM NaH 2

PO

4 .2H 2 0, 120 mM NaCI), pH 7.0 at 32 0 C for 18 hr in the dark. After a 10 min wash with 20 ml water per cornea, a modified silver stain to stain endothelial cell boundaries was performed by application of 1% w/v AgNO 3 for 1 min and subsequent exposure to light The endothelium was surgically removed using a 23 gauge needle and toothed forceps, and mounted in Kaiser's glycerol jelly (12.5% w/v gelatin, 87.5% v/v glycerin) on chrome-alum subbed slides. To detect E. coli 0galactosidase in 293 cells, cells were washed twice with PBS, fixed on ice with 0.25% gluta.aldehyde in PBS for 5 min and washed twice with ice-cold PBS. Staining was then performed as described above.

Quantification oflacZ expression. To quantify the number of cells expressing the reporter gene, corneal endothelial flat-mounts were examined by light microscopy and photographed on 35 mm slide film at standard magnifications. The slides were projected at a standard distance and magnification. Total numbers of endothelial cells and lacZ positive cells were counted within frames of known dimension. For each cornea, three areas on each of two different slides taken of representative areas of the flat-mount were counted, and the mean and standard deviation (SD) calculated.

Adenoviral-mediated reporter gene transfer to ovine corneal endothelium. Ovine corneas were transfected in vitro with Ad-lacZ. At 6.6x10 2 6.6x10 4 pfu/corea, single 3galactosidase-positive cells were observed scattered over the endothelial monolayer.

CATEMPornc. p2doc.ll/IWA) 17- Increasing the virus concentration increased the number of P-galactosidase-positive cells to a maximum of approximately 50% (Fig. 2A), although a drop in expression was observed at 6.6x10 8 pfu/cornea. A concentration of 6.6x10 6 7 pfu/comea was judged to yield optimal expression. None of the negative controls (no virus applied, Ad-mock applied) showed expression of P-galactosidase at any time. Reporter gene expression was observed only in corneal endothelium, not in stromal keratocytes. No visible toxic effects on the cornea were observed at any virus concentration. The influence of varying the time that the vector was in contact with corneal endothelium was investigated at 6.6x10 6 and 6.6xl0 7 pfu/cornea (Fig. 2B): about 30% of cells were infected within the first hour, the number of positive cells increasing to about 50% at 2 hours. Duration of reporter gene expression was examined in a time-course experiment using 6.6x107 pfu/cornea and an infection time of 1.5 hr: 30% cells expressed p-galactosidase after 24 hr, rising to approximately 70% at day 6, and expression remained at this level for the 28-day observation period (Fig. 2C).

Replication-deficient adenoviruses, which remain episomal and do not integrate into the host genome, are suitable vectors for gene therapy of amitotic cells. A replicationdefective adenovirus proved an efficient vector for gene transfer to 70-80% of ovine corneal endothelial cells. Optimal expression of the reporter gene in vitro was obtained with 6.6 x 10 6 pfu per cornea. Given that the sheep corea contains approximately 8 x 10 endothelial cells, 6.6 x 106 pfu represents a multiplicity of infection of >10 virions per cell.

Infection at higher concentrations of the vector was less efficient, but no obvious toxic effects were apparent at any viral concentration. The optimal concentration was similar to that found by other authors for infection of rabbit corneal endothelium with adenoviral vectors (13-15). Other authors have observed reporter gene expression in 7% of rabbit corneal endothelial cells using Lipofectamine and in a relatively small proportion of bovine endothelial cells using dioleoyl phosphatidylethylanolamine More recently, George and his colleagues have demonstrated that activated polyamidoamine dendrimers, a novel class of non-viral agent, can successfully be used to transfer a gene into 6-10% of rabbit and human corneal endothelial cells Adenovirus, however, appears significantly more efficient in achieving gene transfer than are non-viral agents.

C \TEMP\comol tp2 doc-I III/O1I -18- Detection of IL-10 mRNA in transfected ovine corneas. Fresh corneas prepared as described above were infected with 4.5x10 6 pfu Ad-mock or Ad-IL-10 for 2 hr or were incubated in medium without viral vector. They were then incubated in 3 ml complete medium at 32 0 C in air for 24 hr, after which a further 2 ml of complete medium was added and organ-culture was continued. At various time points thereafter, a central 8 mm diameter full-thickness disc of cornea was trephined and snap-frozen in liquid nitrogen.

Each disc was pulverised in a pre-chilled stainless steel mortar and pestle. Total RNA was extracted with Total RNA Extraction Reagent (Advanced Biotechnologies Ltd., Surrey, UK), treated with DNAse (GlassMax MicroIsolation Kit, Life Technologies, Melbourne, VIC, Australia) and reverse-transcribed using a commercially-available first-strand cDNA synthesis kit (Amersham Pharmacia Biotech UK Limited, Buckinghamshire, England) according to the manufacturers' recommendations. To control for residual ovine genomic or viral DNA contamination, samples were subjected to the same reverse-transcription step after inactivation of the reverse transcriptase at 95 0 C for 60 min. Dilutions of cDNA were amplified in 25 t1 total volume by PCR. The reaction mixture for IL-10 and p-actin was mM Tris-HCL (pH 0.15M KCI (Perkin Elmer Roche Molecular Systems, Branchburg, New Jersey, USA), 0.2 mM of each dNTP (Amersham Pharmacia Biotech UK Limited, Buckinghamshire, England), 1.5 mM MgC12, 1 mM of each primer, 1 unit AmpliTaq-Gold (all from Perkin Elmer Roche Molecular Systems, Branchburg, New Jersey, USA) and 5 p.1 of sample. The reaction buffer for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) contained 2 mM MgCl 2 but was otherwise identical. Primer sequences amplified a 307 base pair region for IL-10 GCAGCTGTACCCACTTCCCA-3' [SEQ ID NO 5'-AGAAAACGATGACAGCG-3' [SEQ ID NO a 317 base pair region for p-actin 3',[SEQ ID NO 3] 5'-CATCTCTTGCTCGAAGTCCA-3'[SEQ ID NO 4] and a 527 base pair region for GAPDH (5'-ACCACCATGGAGAAGGCTGG-3'[SEQ ID NO CTCAGTGTAGCCCAGGATGC-3'[SEQ ID NO After one cycle of 15 min at 94C, cycles of amplification were performed, each consisting of annealing at 55 0 C for sec, extension at 72 0 C for 30 sec and 94 0 C for 1 min, final extension at 55 0 C for 30 sec, C \TEMPVollt.;i Ip 2 doc-l ll/Hil 19- 72 0 C for 20 sec and 35 0 C for 10 sec. Amplified products were electrophoresed on agarose w/v gels.

Orthotopic corneal transplantation in sheep. Adult female Merino-cross breed sheep were acclimatised in groups of at least two animals for at least one week in indoor pens and were fed water ad libitum and chaff supplemented with lucerne hay. Twelve mm diameter penetrating corneal transplantation was performed as previously described in the right eye only. Post-operative care and inspection were as previously described and every graft was examined at the slit-lamp each day. Groups of sheep received unmodified corneal grafts, corneas infected with Ad-mock, or corneas that had been infected with Ad-IL-10 or Ad-P40-IL-12 according to optimised procedures. The order in which sheep were grafted was random amongst all groups. Rejection was defined as reported previously In several sheep with long-surviving corneal grafts, attempts were made to induce rejection by placement of 8-0 braided silk sutures into the graft under general anaesthetic, as an inflammatory stimulus. Approval for all experimentation was obtained from the institutional Animal Welfare Committee.

End-point histology of corneal allografts. Corneal tissue was fixed in buffered formalin, embedded in paraffin wax, cut at 8 lm and stained with haematoxylin and eosin.

Immunoperoxidase staining of corneal allografts. Hybridoma culture supernatants containing mouse mAbs to sheep cell-surface determinants were obtained from the Department of Veterinary Science, University of Melbourne, Parkville, VIC, Australia and included: SBU 41.19, anti-MHC class 1 monomorphic epitope SBU 28.1, anti-MHC class II monomorphic epitope SBU 1-11-32, (unrestricted) antigen SBU 44.38, anti-CD4 and SBU 38.65, anti-CD8 (26, 27); SBU 20.27, anti-CD1 and SBU 72.87, anti-CD1 la/LFA-1 Culture supematants from the hybridomas P3X63Ag8 (IgGI isotype; European Collection of Animal Cell Cultures, Porton Down, Wiltshire, UK) and SAL5 (IgG2a isotype; gift of Dr L Ashman, IMVS, Adelaide, SA, Australia) were used as negative controls. Grafted eyes were harvested C \TEMPcom.l Ip2 doc.l I I llsll C immediately post-mortem and the cornea excised, fixed, stained and scored as previously N described Adenoviral antibody titres influidsfrom sheep with corneal allografts. Immediately post- 0 mortem, anterior chamber fluid was collected and snap-frozen at -80 0 C. Venous peripheral blood was also collected, the serum separated and similarly snap-frozen.

D Antibody titres to adenovirus were determined by a standard complement fixation test in O the local reference laboratory using reagents from Biowhittaker Northfield Laboratories, Adelaide, SA, Australia.

Statistical analysis of data. Corneal graft survival data were analysed with the Mann- Whitney U-test, corrected for ties.

Detection ofIL-10 mRNA in IL-10 gene-modified organ-cultured ovine corneas. was used to transfer the gene encoding ovine IL-10 into sheep corneal endothelium using conditions optimised for reporter gene expression, and the corneas were cultured in vitro for up to 21 days. Reverse transcription PCR was used to detect presence of mRNA for p-actin and GAPDH served as housekeeping controls. No amplification of genomic or adenoviral IL-10 was observed in controls in which the reverse transcriptase had been inactivated after DNAse-I treatment of the isolated RNA preparations. Specific mRNA for ovine IL-10 was observed 24 hr after adenoviral infection and at various time points thereafter (Table and could still be detected after corneas had been organ-cultured for 21 days (Fig. 3).

Orthotopic transplantation of gene-modified donor corneas in outbred sheep. The Ad-ILand Ad-P40-IL-12 vectors were used to infect corneal endothelium of donor corneas immediately prior to orthotopic comeal transplantation in outbred sheep (Fig. Controls included unmodified donor corneas and corneas infected with Ad-mock. Corneal graft survival data are shown in Table 2: corneal grafts modified by insertion of the gene encoding IL-10 into the donor endothelium survived significantly longer than unmodified controls (p 0.019) or the combined unmodified and mock virus-infected control groups C \TEMPcorcil Ip2 doc- I I/II /1 S-21- (p 0.011). There was no difference in the time at which host vessels crossed the grafthost-junction amongst the groups (p 0.05). Longer survival was also demonstrated in corneas modified by c-section of the genes encoding the P-40 subunit of IL-12 (median days). Post-operative inflammation was no more severe, and lasted for no longer, in the O 5 groups receiving adenovirus-treated corneas compared with the controls. End-point 0histology in sheep that showed clinical rejection of their grafts showed a similar picture in Sall instances: there was no difference amongst the experimental groups. Similarly, immunoperoxidase staining showed that rejecting gene-modified corneas contained a cellular infiltrate similar to that seen in rejecting unmodified or mock virus-infected grafts, with a substantial infiltrate of both CD4-positive and CD8-positive cells. No antibody to adenovirus was detectable in anterior chamber fluid or serum from 2 sheep at post-mortem.

Attempts were made to induce rejection in two sheep from the IL-10 group with genemodified, long-surviving (>150 days) corneal grafts by placement of silk sutures into the graft at 196 and 303 days post-graft, respectively. In both cases, inflammation of the graft ensued and rejection occurred within two weeks, indicating that neither recipient was tolerant of the graft. Immunohistochemistry indicated that the infiltrate in these rejected grafts was similar to that seen in unmodified grafts.

Adenovirus binds to surface receptors and enters the cell by endocytosis via clathrincoated vesicles, a fast process (36, 37). DNA replication in replicative adenoviruses starts approximately 8 hours after infection In the sheep cornea, most adenoviral infection occurred within the first hour, but a delay of 5-6 days was observed before reporter gene expression was maximal. A time lag of 3-7 days for lacZ expression driven by either the CMV or RSV promoter has been observed after adenoviral transfer to human corneal endothelium (12, 14, 15). The inventors observed lacZ expression in the sheep corneal endothelium to be stable for 4 weeks in vitro. Investigators working in the rabbit have found expression oflacZ for 3-4 weeks in in vitro experiments but for only 1-2 weeks after orthotopic corneal transplantation The protein p-galactosidase has a half-life of 2 weeks in neurons (39) but shorter expression has been observed in other tissues such as respiratory epithelium possibly due to gene silencing by promoter extinction In C ITEMPtomcl Ip2 do- I /IIIII -22the absence of a monoclonal antibody specific for ovine IL-10, expression of product in ovine comeal endothelial cells was assessed indirectly by detection of mRNA for IL-10 in transfected, organ-cultured corneas. The inventors were able to detect mRNA in ovine corneal endothelium for at least 3 weeks in vitro.

Obvious toxicity that could be attributed to the use of the adenoviral vector was absent.

The adenoviral construct used to deliver the target gene to donor corneal endothelium did not elicit a measurable antibody response in the sheep after corneal transplantation, and did not induce noticeable ocular inflammation over the time-course of the experiment. Host vessels extended from the limbus towards all corneal grafts at the same rate, irrespective of the experimental group. In most animals that received gene-modified donor corneas, neovascularization was not accompanied by corneal graft rejection and the comeal vessels in these sheep did not maintain patency.

In these experiments, gene transfer of IL-10 to the donor cornea immediately prior to transplantation prolonged corneal allograft survival to a significant extent in the cohort as a whole. In two cases, graft survival was prolonged indefinitely 150 days). Allograft survival was also prolonged in the group where corneal tissue was modified with P-40 of IL-12. It is notable that these result were obtained without the use of any other immunosuppressive therapy at all and in particular, without use of topical glucocorticosteroid..

That tolerance was not induced in the long-survivors is evinced by the observation that these animals did reject their grafts after deliberate application of an inflammatory stimulus to the graft.

Example 2 Two human corneas retrieved from the one donor by the Eye Bank of South Australia were obtained, following consent from the donor's family according to standard protocols and with permission from the institutional human ethics committee.

C\TF.MP'cortca] p2 doc.- 11/10/) -23- These corneas were prepared for infection with an adenoviral vector in the same way as are sheep corneas as described above (that is, excised from the eye with a 1-2 mm scleral rim).

They were infected with El, E3 replication-defective adenovirus type 5, with cDNA encoding green fluorescent protein (GFP) in the vector expression cassette.

Infection conditions were exactly the same as those optimised for the viral vectors above and sheep corneas. Corneas were organ-cultured in the same way as the sheep corneas (that is, in the same culture vessel and with the same medium).

The corneas were harvested after 5 days of culture, fixed and examined under the fluorescent microscope. The corneal endothelial monolayers clearly showed a small number of cells expressing GFP, as assessed by bright apple green fluorescence in the nucleus and cytoplasm of the positive endothelial cells.

The present invention has been described by way of example only and it should be recognised that modifications and/or alterations to the specific aspects of the invention which would be apparent to persons skilled in the art based on the disclosure herein, are also considered to fall within the spirit and scope of the invention.

C \TEMIcorncl Ip2 doc- llI/11 I1 -24- TABLE 1. Detection of mRNAs for ovine IL-10 or housekeeping genes P-actin and GAPDH in ovine corneas after infection with adenoviral vectors a Time after Primer Corneas infected with:a infection identity medium control Ad-mockb 2 hours IL-10 d P-actin/GAPDH +d 3 days IL-1 P-actin/GAPDH 7 days IL-10 P-actin/GAPDH days IL-10 NTe p-actin/GAPDH NT 14 days IL-10 NT P-actin/GAPDH NT 21 days IL-10 NT P-actin/GAPDH NT a For each primer at each time-point, 1-3 individual corneas were examined; b Ad-mock, replication deficient El, E3-deleted adenovirus type 5 containing an empty plasmid; r Adreplication deficient El, E3-deleted adenovirus type 5 encoding full-length ovine d represents no signal detected by PCR, represents a weak positive signal detectable only in an undiluted cDNA sample, represents a strong positive signal; e NT, not tested.

C \Tl MPorc.l Ip 2 duo-l/l nll1

INO

Table 2. Survival of control and gene-modified orthotopic corneal grafts in outbred sheep transplanted with unmodified donor corneas, or with corneas transfected before transplantation with Ad-mock, or with corneas transfected before transplantation with Ad- Ad-P40-IL-12 or Ad-Il-4 Donor cornea n Day vessels Day of rejection crossed into grafta Unmodified 7 11,10,9,8,10,9,9 18,19,19,20,20,22,32 median 9 median Mock-transfected 3 5,7,8 19,21,29 median 7 median 21 9 5,9,9,10,10,9,11,11,9 19,20,30,33,55,66,88, median 9 >196,>300 b median L-10 transfected 9 22,23,32,36,45,>50,93,93, >100 median a For each recipient sheep, the day post-graft at which corneal blood vessels crossed from the recipient corneal edge into the graft is shown, together with the day post-graft at which the graft was deemed to have undergone rejection. Individual recipients in columns 3 and 4 are listed in the same order; bp 0.019 compared with unmodified controls, p 0.011 compared with combined control groups (Mann-Whitney test, two-tailed).

C \TEMjcollc.dl ip 2 doc-I I,/Iln/ -26-

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Claims (34)

1. A method of modifying cells of corneal tissue to express an active agent comprising exposing harvested corneal tissue such that said cells produce an active agent, said method comprising introducing into said cells an effective amount of an expression C vector which comprises a nucleotide sequence encoding said active agent and culturing Ssaid cells for a time and under conditions sufficient for said cells of said corneal tissue C, to express said nucleotide sequence and produce said active agent such that cells of said corneal tissue will express the active agent and prolong corneal graft survival, improve corneal graft healing or prevent corneal tissue rejection as well as for the provision of therapy for ocular infection, wounds, fibrosis or other ocular disorders.

2. A method of improving corneal graft healing and/or prolonging graft survival comprising modifying cells of corneal tissue such that said cells produce an active agent, said method comprising introducing into said cells an effective amount of an expression vector which comprises a nucleotide sequence encoding said active agent and culturing said cells for a time and under conditions sufficient for said cells of said corneal tissue to express said nucleotide sequence and produce said active agent, .and then transplanting the corneal tissue to an eye of a recipient.

3. A method according to claim 1 or 2 wherein the active agent is a peptide hormone, a cytokine or an analogue thereof.

4. A method according to claim 3 wherein the cytokine is an interleukin, an interferon or a growth factor, or an analogue thereof. A method according to claim 3 wherein the cytokine is selected from IL-10, the component of IL- 12, Bcl2, interferon y, interferon a and TGF P.

6. A method according to claim 3 wherein the cytokine is the P-40 component of IL-12 006W C \rEMPNoc.,l ip: do(- Iltl -32-

7. A method according to claim I or 2 wherein the active agent has immunomodulatory, anti-inflammatory, anti infective, tissue regeneration, wound healing, anti-apoptotic, or fibrosis reduction activity

8. A method according to claim 1 to for treatment of ocular diseases including glaucoma, keratoconus, corneal dystrophies, comeal infections, tumours of the eye, proliferative lesions, pterygium and inflammatory disorders of the eye including Stevens-Johnson syndrome and mucous panphigoid

9. A method according to claim 1 or 2 wherein the corneal tissue is harvested from a mammal. A method according to claim 9 wherein the corneal tissue is harvested from a human.

11. A method according to claim 1 or 2 wherein the corneal tissue is an artificial cornea.

12. A method according to claim 1 or 2 wherein the modified cells are epithelial cells, stroma cells or endothelial cells.

13. A method according to claim 12 wherein the modified cells are endothelial cells.

14. A method according to claim 1 or 2 wherein the expression vector is a viral, bacterial or plasmid vector.

15. A method according to claim 14 wherein the expression vector is an adeno-associated viral vector or an adenoviral vector.

16. A method according to claim 1 or 2 wherein the expression vector comprises a liposome.

17. A method according to claim 1 or 2 wherein the expression vector comprises C \TEMP o-rnclI ip doL. I /111 \O (N -33- nucleotide sequences encoding for two or more active agents.

18. A corneal tissue comprising cells modified to express a nucleotide sequence to produce an active agent, said nucleotide sequence is not expressed in normal corneal tissue or is expressed at elevated levels relative to normal corneal tissue. IND 0 19. A corneal tissue comprising cells modified to produce an active agent, wherein Cmodification is by exposing harvested corneal tissue to an effective concentration for transfection of an expression vector which comprises a nucleotide sequence encoding for the active agent, for a period and under conditions sufficient to allow transfection. A corneal tissue according to claim 18 or 19 wherein the active agent is a peptide hormone, a cytokine or an analogue thereof.

21. A corneal tissue according to claim 20 wherein the cytokine is an interleukin, an interferon or a growth factor, or an analogue thereof.

22. A corneal tissue according to claim 20 wherein the cytokine is selected from IL-10, the component of IL-12, Bcl2, interferon y, interferon a and TGF 3.

23. A corneal tissue according to claim 20 wherein the cytokine is the P-40 component of IL-12

24. A corneal tissue according to claim 18 or 19 wherein the active agent has immunomodulatory, anti-inflammatory, anti infective, tissue regeneration, wound healing, anti-apoptotic, or fibrosis reduction activity. A corneal tissue according to claim 18 or 19 wherein the corneal tissue is harvested from a mammal.

26. A corneal tissue according to claim 25 wherein the corneal tissue is harvested from a F7-4& C \TEMP oincrlIp2 di-ll 11i lI -34- human.

27. A corneal tissue according to claim 18 or 19 wherein the comeal tissue is an artificial cornea.

28. A corneal tissue according to claim 18 or 19 wherein the modified cells are epithelial cells, stroma cells or endothelial cells.

29. A corneal tissue according to claim 28 wherein the modified cells are endothelial cells. A corneal tissue according to claim 19 wherein the expression vector is a viral, bacterial or plasmid vector.

31. A corneal tissue according to claim 30 wherein the expression vector is an adeno- associated viral vector or an adenoviral vector.

32. A corneal tissue according to claim 19 wherein the expression vector comprises a liposome.

33. A corneal tissue according to claim 19 wherein the expression vector comprises nucleotide sequences encoding for two or more active agents.

34. An expression vector for use in in modifying corneal tissue to express a nucleotide sequence to produce an active agent, said nucleotide sequence is not expressed in normal corneal tissue or is expressed at elevated levels relative to normal corneal tissue An expression vector according to claim 34 wherein the active agent is a peptide hormone, a cytokine or an analogue thereof.

36. An expression vector according to claim 34 wherein the cytokine is an interleukin, an interferon or a growth factor, or an analogue thereof. C TEMP.oicalip n I .o I I l

37. An expression vector according to claim 34 wherein the cytokine is selected from IL- the P-40 component of IL-12, Bcl2, interferon y, interferon at and TGF P.

38. An expression vector according to claim 34 wherein the cytokine is the component of IL-12

39. An expression vector according to claim 34 wherein the active agent has immunomodulatory, anti-inflammatory, anti infective, tissue regeneration, wound healing, anti-apoptotic, or fibrosis reduction activity. An expression vector according to claim 34 wherein the expression vector is a viral, bacterial or plasmid vector.

41. An expression vector according to claim 34 wherein the expression vector is an adeno- associated viral vector or an adenoviral vector.

42. An expression vector according to claim 34 wherein the expression vector comprises a liposome. An expression vector according to claim 34 comprising nucleotide sequences encoding for two or more active agents. DATED this 12th day of May, 2006 Douglas John Coster AND Sonja Klebe AND Keryn Anne Williams By DAVIES COLLISON CAVE Patent Attorneys for the Applicant