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M. Siddique, S. E. Barker, R. R. Ali, S. J. Ono; In-vivo Expression of Green Fluorescent Protein in the Allergic Conjunctiva - A Model for Gene Therapy. Invest. Ophthalmol. Vis. Sci. 2007;48(13):200.
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Current therapies for allergic eye disease require high doses, frequent application, give rise to systemic side effects, and tendency for poor patient compliance. Gene therapy offers a novel approach to treat chronic diseases by directly delivering the genes encoding for anti-inflammatory cytokine proteins for sustained therapeutic effect.
To evaluate the feasibility of developing gene therapy for the treatment of allergic eye disease. To study the in vivo efficiency of GFP gene using lentiviral gene delivery vectors to the conjunctiva of normal and allergic mouse.
Lentivirus- GFP construct was produced by transient transfection and pseudotyped them with the vesicular stomatitis virus glycoprotein G envelop in human embryonic kidney (HEK) 293 T cell line. The construct was injected subconjunctivally into two groups of A/J naïve and allergic mice of 12 weeks age. Allergic model of mice was prepared by immunization with Short ragweed (SRW) pollen and alum for 5 weeks. We determine the optimal dose, site of injection and maximum conjunctival gene expression in pilot experiments. Signs of inflammation and toxicity in and around the eyes were evaluated by slit lamp observation. At day 4, 7, 14, 21 and 28 post injections, eyes in both groups were enucleated and embedded in optimum cutting temperature (OCT) compound. Sections stained with Propidium Iodide in fluorescent mounting media for visualizing of GFP expression and distribution.
A single dose of 8µl Lentivirus- GFP construct considered to be sufficient to cause transduction of GFP in the conjunctiva. GFP expression was seen in the bulbar and forniceal conjunctiva at day 4 and lasted up to 21 days. The GFP gene expression in the allergic group was similar to the expression in the naïve group. There was no expression at day 28 in both groups. H & E staining of the sections show no morphological changes with the viral construct. The cornea remained clear and no signs of inflammation in the eye were present during the course of the experiment.
Conjunctiva can be targeted for gene therapy and a single dose of the desired cytokine can be delivered through the conjunctiva for therapeutic effects. Also inflammatory response does not affect expression of therapeutic gene and lentiviral mediated gene therapy can be safely used. The results provide preliminary data and potential for in vivo gene therapy in the conjunctiva for external eye disorders.
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