Ocular gene therapy
a gene therapy and gene technology, applied in the field of ocular gene therapy, can solve the problem that the transgenic technique is not directly applicable to human therapy
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example 1
Delivery of Exogeneous Nucleic Acid to Corneal Epithelial Cells
[0058] A β-galactosidase-expressing recombinant adenoviral vector was constructed by first building a shuttle plasmid, in which the CMV / β galactosidase expression cassette was flanked by adenovirus DNA sequences, such that when cotransfected with the adenoviral deletion mutant DNA d1324 into 293 cells, a homologous recombination event placed the β-galactosidase expression cassette into the E1 region of the virus. The resulting E1-deficient recombinant virus was thus replication incompetent in all cells but 293 cells, and expressed the E. coli β-galactosidase gene. This gene product may be assayed for its enzyme function, or assayed histochemically by exposing the enzyme to a substrate, X-gal, resulting in a characteristic blue stain in the cells that are expressing the marker gene.
[0059] The recombinant, replication-incompetent viral vectors were produced in 293 cells, a human fetal kidney cell line, which expressed th...
example 2
Delivery of Exogenous Nucleic Acid to Corneal Endothelial Cells
[0065] 20 μl of aqueous humor from the anterior chamber of anesthetized rats was removed using a 30 g needle and a Hamilton syringe. This fluid was replaced with 20 μl of a 1×109 pfu / ml solution of the replication-incompetent recombinant adenoviral vector described above which delivered a β-galactosidase marker gene. Control rats were treated in the same way, but received vehicle alone. 24 hours later the animals were sacrificed, and the eyes were mounted in Tissue Tek, frozen, sectioned and stained for β-galactosidase protein. As shown in FIGS. 2A and 2B, there was clear positive staining of the majority of cells lining the posterior surface of the cornea (the corneal endothelial cells) in the animals that received the viral vector, but no staining in the animals that received vehicle alone. Furthermore, there was also staining of the cells of the ciliary body epithelium in animals that received vector.
example 3
Delivery of Exogeneous Nucleic Acid to Choroid Ocular Cells
[0066] 5 μl of a 1×10 9 pfu / ml solution of a β-galactosidase expressing recombinant adenoviral vector was injected into the vitreous humor (posterior segment) of the eye in anesthetized rats. Control rats were treated in the same way, but received vehicle alone. 24 hours later the animals were sacrificed, and the eyes were mounted in Tissue Tek, frozen, sectioned and stained for β-galactosidase protein. There was clear positive staining of some of the cells of the choroid, which is the vascular coat surrounding the posterior part of the eye. There was no such staining in animals that received vehicle alone.
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