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Abstract #8164 Published in IGR 4-3
Preservation of aqueous outflow facility after second-generation FIV vector-mediated expression of marker genes in anterior segments of human eyes
Loewen N; Bahler C; Teo W; Whitwam T; Peretz M; Xu R; Fautsch MP; Johnson DH; Poeschla EMInvestigative Ophthalmology and Visual Science 2002; 43: 3686-3690
PURPOSE: Feline immunodeficiency virus (FIV)-based lentiviral vectors produce effective genetic modification of the trabecular meshwork (TM) of human eyes in organ-perfusion culture, resulting in high-level expression of a β-galactosidase marker gene (lacZ) without loss of TM cellularity or architecture. However, effects on aqueous outflow physiology have not been determined, and the ability to monitor FIV vector transgene expression in living TM in situ has not been established. In the current study, transgene expression and outflow facility were evaluated in perfused human anterior segments after FIV vector transduction of lacZ or of a marker gene that can be monitored noninvasively, enhanced green fluorescent protein (eGFP). METHODS: Second-generation FIV vectors were made with a protocol for scaled-up production that requires ten times less input DNA and allows simplified concentration. One vector encodes β-galactosidase (vector CT26), and the other (bicistronic) encodes eGFP and neomycin phosphotransferase (vector GiNWF). Three pairs of eyes were injected with 1 x 108 transducing units (TU) of CT26 in the right eye and with a control (mock lacZ) vector in the left eye. Three others were injected with 1 x 108 TU GiNWF in the right eye only, with the left eye serving as an uninjected control. Intraocular pressure was recorded and transduction efficiency was determined. RESULTS: The modified protocol produced high-titer FIV vectors, and coordinate expression of marker genes was observed with the bicistronic vector. In human eyes, the eGFP and lacZ vectors transduced 79 ± 15% and 82 ± 4% of TM cells, respectively, without cell loss compared with control eyes. Transduction and marker gene expression caused a transient decrease of outflow facility (30 ± 22%, P = 0.02), which resolved after 48-72 hours. CONCLUSIONS: FIV vectors produce high-level expression of eGFP in the TM of the cultured human eye, with transduction efficiency similar to that obtained with β-galactosidase vectors. Transduction and expression of these marker genes results in small and transient changes in outflow facility, suggesting suitability of this class of vectors for glaucoma gene therapy.
N. Loewen, MD, Molecular Medicine Program Mayo Clinic, Rochester, MN, USA
Classification:
3 Laboratory methods
2.5 Meshwork (Part of: 2 Anatomical structures in glaucoma)
