Purchase this article with an account.
N.A. Loewen, W. Teo, M.P. Fautsch, C.K. Bahler, D.H. Johnson, E.M. Poeschla; Transgene-Specific Toxicity in Lentiviral Vector-Transduced Feline TM In Vivo . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1147.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: Monitoring of transgene expression in live animals, which is facilitated by GFPs, is desirable for glaucoma gene therapy experimentation. In explanted human eyes, expression of eGFP or of ß-galactosidase (ß-gal) with FIV vectors is associated with minor and transient outflow facility changes despite excellent gene transfer and marker protein over-expression in TM. Here we assessed 3 different marker transgenes in vivo. Methods: Nine domestic cats received anterior chamber (AC) bolus injections of transducing unit (TU)-normalized, VSV-G pseudotyped FIV vectors CT26 (ß-gal), GINWF (eGFP from Aquorea victoria) or RGWF (GFP from Renilla reniformis). Two were injected with 10^8 TU GINWF into the right eye (R), while the left (L) served as an uninjected control. Four received GINWF (R) and CT26 (R) (10^8 and 10^7 TU in two cats each). Three additional cats were injected with 10^8 TU RGWF (R) and 10^8 TU GINWF (L). Results: Expression plateaued at 17±9 days and was confined to the TM except for a few cells in the iris epithelium. High expression was observed for more than 52 days with Aequoria eGFP, which produced brilliant fluorescence throughout the TM but also resulted in subsequent iritis and a 5.7±4.3 mmHg IOP decrease, followed by termination of eGFP expression several days later. In contrast, loss of expression was not seen with ß-gal expression, which persisted and was highly expressed throughout the TM at sacrifice. Systemic and local corticosteroids did not prevent GFP-induced iritis. Iritis was clinically minimal with Renilla GFP, although expression was less. Histologically, Aequoria eGFP- and, to a lesser extent, Renilla GFP-transduced TMs, had lymphocytic infiltrates, while ß-gal-transduced TMs did not. In vitro, sera from injected animals had low to moderate neutralizing activity (1:100-1:1000 dilutions) against both VSV-G- and control lymphocytic choriomeningitis virus (LCMV) envelope-pseudotyped FIV (and HIV-1) vectors, but eGFP- or VSV-G-specific antibodies were not detected. Conclusions: Aequoria eGFP and Renilla GFP induced clinically and histologically evident AC inflammatory responses that were associated with termination of expression. In contrast, ß-gal did not. Since these initial studies revealing marker protein-specific toxicity involved maximum over-expression in highly transduced TMs, dose response studies are needed to understand the implications.
This PDF is available to Subscribers Only