Long-term retinal transgene expression with FIV versus adenoviralvectors

[摘要] Purpose: Gene therapy for chronic retinal diseases will requirelong-term expression of therapeutic transgenes. Lentiviral andadenoviral (Ad) vectors are gene delivery systems with markedlydifferent properties. Lentiviral vectors require integration into thehost genome, which facilitates long-term expression, while Ad vectorsremain episomal. We compared time course, location, and extent oftransgene expression from replication-deficient feline immunodeficiencyvirus (FIV) vectors and Ad vectors in neonatal rat retina.Methods: A dose-response study was conducted to determine theoptimal subretinal dose for comparison of FIV and Ad vectors with aninternal cassette expressing β-galactosidase under transcriptionalcontrol of the CMV immediate-early gene promoter/enhancer. Forty-twofive-day old Sprague-Dawley rats received subretinal injections of 2 μl containing 2x103 transducing units (TU, n=14), 2x104 TU(n=14) or 2x105 TU (n=14) of FIV vector (right eye) and Ad vector(left eye). Expression was evaluated 48 h after transduction. In thesubsequent long-term expression study, 60 five-day old rats received asubretinal injection of 2x105 TU FIV vector (right eye) and Advector (left eye). Ten pairs of eyes were analyzed at 1 week, 1 month, 3months, 6 months, 12 months, and the remainder at 16 months. Eye cupswere evaluated in a masked manner for extent of β-galactosidaseexpression (graded 0-5) by whole mount microscopy and by cross sectionalhistology.Results: In the dose-response study, 2x105 TU resulted inconsistent, widespread retinal transduction with both vectors and wasselected as the dose for the subsequent study. In the long-termexpression study, FIV vector resulted in a higher grade of expressionthan Ad at multiple single time points and produced higher overallexpression when data from all eyes across the entire 16 month study wereanalyzed (p=0.01). Retinal expression was present at 16 months with bothvectors. β-galactosidase expression was limited to the retinalpigment epithelium (RPE) until the first month, but later was also foundto a lesser extent in neurosensory retina with each vector. In contrastto FIV, most Ad injected eyes showed signs of focal accumulation ofmacrophage-like cells with disrupted retinal architecture.Conclusions: Both FIV and Ad vectors result in long-term transgeneexpression in RPE after subretinal injection. FIV vectors show morepromise than Ad as delivery systems for retinal diseases since theytransduce greater areas of RPE, result in less cellular infiltrate, andcause less disruption of retinal architecture. The persistent expressionat 16 months of follow-up suggests that these lentiviral vectors areuseful for gene therapy of chronic retinal diseases.

[学科分类] 生物化学/生物物理