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P.D. Khare, W.–L. Teo, N. Loewen, R. Barraza, M.P. Fautsch, D.H. Johnson, E.M. Poeschla; Long–Term Genetic Modification of Trabecular Meshwork by Lentiviral Vectors: An 18 Cat Study . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4770.
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Lentiviral vectors may be useful for glaucoma gene therapy. We present results of transcorneal lentiviral vector injections in 18 domestic cats. Animals were followed long–term (1.2 to 2.2 years). Non–invasive in vivo and detailed post–mortem examinations were done.
18 domestic cats received single bolus anterior chamber injections of FIV vector in doses of 106 (16 eyes) or 107 (19 eyes) transducing units (TU). Vectors encoded either eGFP, or a combination of feline myocilin (fMYOC or fMYOC with a Y423H mutation, equivalent to human MYOC Y437H) plus eGFP. The latter was a mono–cistronic dual gene arrangement [5' to 3': fMYOC cDNA, internal ribosome entry site (IRES), eGFP cDNA]. A control vector lacked a gene in position 1. Long–term follow–up included IOP measurements, slit lamp biomicroscopy, and gonioscopy. Eyes were then enucleated examined with fluorescence microscopy, immunostaining for fMYOC and eGFP, and quantitative real–time vector PCR for vector DNA.
In tissue culture, IRES–mediated eGFP translation was useful but always resulted in much less eGFP expression per vector–transduced cell than eGFP alone or eGFP in the first gene position. In vivo, single gene eGFP vectors resulted in long–term, high–grade eGFP expression detectable non–invasively for over two years. For IRES–translated eGFP, expression was detected in 29 of 32 eyes by gonioscope but was less than seen with translation initiated at the 5' cap site. However, considerably more extensive GFP expression was discovered when these TMs were examined as dissected flat mounts after sacrifice. Expression of myocilins and eGFP was further confirmed by immunohistochemistry. Vector DNA copy number correlated with transgene expression. Mild AC inflammation was observed in some animals but resolved after 1–2 weeks. In all animals, IOP at the end of the study did not differ significantly from initial IOP (19–25 mmHg).
Sustained, high–grade eGFP expression lasting over two years was achieved in TM with lentiviral vectors. Vector–mediated eGFP fluorescence can be detected non–invasively, even when incorporated as a secondary, IRES–translated marker. Non–invasive monitoring is helpful but underestimates long–term in vivo transgene expression, since considerably more eGFP expression was discovered after enucleation. Transduction of fMYOC or Y423H fMYOC did not cause elevated IOP in this study.
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