Corneal cells expressing active agents and methods of use thereof

Abstract

The invention relates to methods of modifying cells of corneal tissue to express an active agent, to modified corneal tissue, to vectors utilized in such methods and to methods of xeno- and allo-transplantation utilizing the modified corneal tissue. The method of modifying cells of corneal tissue to express an active agent involves 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 sufficient to allow infection, such that cells of said corneal tissue will express the active agent.

What is claimed is:  
     
         1 . A method of modifying cells of corneal tissue to express an active agent comprising 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 sufficient to allow transfection, such that cells of said corneal tissue will express the active agent.  
     
     
         2 . The method according to  claim 1 , wherein the nucleotide sequence consists of DNA.  
     
     
         3 . The method according to  claim 1 , wherein the active agent is selected from the group consisting of a peptide hormone, a cytokine and an analogue thereof.  
     
     
         4 . The method according to  claim 1 , wherein the active agent is selected from the group consisting of an interleukin, an interferon, a growth factor and an analogue thereof.  
     
     
         5 . The method according to  claim 3 , wherein the cytokine is selected from the group consisting of IL-10, IL-4, the P-40 component of IL-12, Bc12, interferon Gamma, interferon Alpha and TGF Beta.  
     
     
         6 . The method according to  claim 1 , wherein the corneal tissue is harvested from a mammal.  
     
     
         7 . The method according to  claim 6 , wherein the mammal is a human.  
     
     
         8 . The method according to  claim 1 , wherein the corneal tissue cells modified are selected from the group consisting of epithelial cells, stroma cells, endothelial cells and combinations thereof.  
     
     
         9 . The method according to  claim 1 , wherein the corneal cells modified are endothelial cells.  
     
     
         10 . The method according to  claim 9 , wherein at least 5% of a sample of corneal endothelial cells are modified.  
     
     
         11 . The method according to  claim 9 , wherein at least 10% of a sample of corneal endothelial cells are modified.  
     
     
         12 . The method according to  claim 9 , wherein at least 20% of a sample of corneal endothelial cells are modified.  
     
     
         13 . The method according to  claim 9 , wherein at least 30% of a sample of corneal endothelial cells are modified.  
     
     
         14 . The method according to  claim 9 , wherein at least 50% of a sample of corneal endothelial cells are modified.  
     
     
         15 . The method according to  claim 9 , wherein at least 70% of a sample of corneal endothelial cells are modified.  
     
     
         16 . The method according to  claim 1 , wherein the effective concentration for infection is between about 1×10 5  to 1×10 10  particle forming units (PFU) per cornea.  
     
     
         17 . The method according to  claim 1 , wherein the effective concentration for infection is between about 5×10 5  to 5×10 8  PFU per cornea.  
     
     
         18 . The method according to  claim 1 , wherein the effective concentration for infection is between about 2×10 6  and about 9×10 7  PFU per cornea.  
     
     
         19 . The method according to  claim 1 , wherein the period sufficient to allow infection is between about 1 minute and about 48 hours.  
     
     
         20 . The method according to  claim 1 , wherein the period sufficient to allow infection is between about 10 minutes and about 24 hours.  
     
     
         21 . The method according to  claim 1 , wherein the period sufficient to allow infection is between about 30 minutes and about 6 hours.  
     
     
         22 . The method according to  claim 1 , wherein the period sufficient to allow infection is between about 1 hour and about 3 hours.  
     
     
         23 . The method according to  claim 1 , wherein the expression vector is selected from the group consisting of a viral, a bacterial and a plasmid vector.  
     
     
         24 . The method according to  claim 1 , wherein the expression vector is an adeno-associated viral vector or an adenoviral vector.  
     
     
         25 . A corneal tissue comprising cells modified to express an active agent which is not expressed by normal corneal tissue or which following modification is expressed at elevated levels relative to normal corneal tissue.  
     
     
         26 . The corneal tissue according to  claim 25 , wherein the active agent is selected from the group consisting of a peptide hormone, a cytokine and an analogue thereof.  
     
     
         27 . The corneal tissue according to  claim 25 , wherein the active agent is selected from the group consisting of an interleukin, an interferon, a growth factor or an analogue thereof.  
     
     
         28 . The corneal tissue according to  claim 25 , wherein the active agent is selected from the group consisting of IL-10, IL-4, the P-40 component of IL-12, PC12, interferon Gamma, interferon Alpha and TGF Beta.  
     
     
         29 . The corneal tissue according to  claim 25 , wherein the cells modified to express an active agent are selected from the group consisting of epithelial cells, stroma cells, endothelial cells and combinations thereof.  
     
     
         30 . The corneal tissue according to  claim 25 , wherein the cells modified to express an active agent are endothelial cells.  
     
     
         31 . A corneal tissue comprising cells modified to express an active agent, wherein modification is by exposing harvested corneal tissue to an effective concentration for infection of an expression vector which comprises a nucleotide sequence encoding for the active agent, for a period sufficient to allow infection.  
     
     
         32 . A corneal tissue comprising cells modified to express an active agent produced according to the method of  claim 1 .  
     
     
         33 . A method of improving corneal graft healing and/or prolonging graft survival comprising exposing harvested corneal tissue to an effective concentration for infection of an expression vector which comprises a nucleotide sequence encoding for an active agent for a period sufficient to allow infection, such that cells of said corneal tissue will express the active agent, and then transplanting the corneal tissue to an eye of a recipient.  
     
     
         34 . An expression vector for use in modifying corneal tissue to express an active agent not expressed by normal corneal tissue or which following modification is expressed at elevated levels relative to normal corneal tissue; the vector comprising a nucleotide sequence encoding for the active agent.  
     
     
         35 . The expression vector according to  claim 34 , wherein the nucleotide sequence is DNA.  
     
     
         36 . The expression vector according to  claim 34 , wherein the active agent is selected from the group consisting of a peptide hormone, a cytokine or an analogue thereof.  
     
     
         37 . The expression vector according to  claim 34 , wherein the active agent is selected from the group consisting of an interleukin, an interferon, a growth factor or an analogue thereof.  
     
     
         38 . The expression vector according to  claim 34 , wherein the cytokine is selected from IL-10, L-4, the P-40 component of IL-12, Bc12, interferon Gamma, interferon Alpha and TGF Beta.  
     
     
         39 . The expression vector according to  claim 34 , selected from the group consisting of a viral, a bacterial or a plasmid vector.  
     
     
         40 . The expression vector according to  claim 34 , wherein the vector is an adeno-associated viral vector or an adenoviral vector.