Ocular localization and transduction by adenoviral vectors are serotype-dependent and can be modified by inclusion of RGD fiber modifications.

To evaluate localization and transgene expression from adenoviral vector of serotypes 5, 35, and 28, ± an RGD motif in the fiber following intravitreal or subretinal administration.Ocular transduction by adenoviral vector serotypes ± RGD was studied in the eyes of mice receiving an intravitreous or subretinal injection. Each serotype expressed a CMV-GFP expression cassette and histological sections of eyes were examined. Transgene expression levels were examined using luciferase (Luc) regulated by the CMV promoter.GFP localization studies revealed that serotypes 5 and 28 given intravitreously transduced corneal endothelial, trabecular, and iris cells. Intravitreous delivery of the unmodified Ad35 serotype transduced only trabecular meshwork cells, but, the modification of the RGD motif into the fiber of the Ad35 viral vector base expanded transduction to corneal endothelial and iris cells. Incorporation of the RGD motif into the fiber knob with deletion of RGD from the penton base did not affect the transduction ability of the Ad5 vector base. Subretinal studies showed that RGD in the Ad5 knob shifted transduction from RPE cells to photoreceptor cells. Using a CMV-Luc expression cassette, intravitreous delivery of all the tested vectors, such as Ad5-, Ad35- and Ad28- resulted in an initial rapid induction of luciferase activity that thereafter declined. Subretinal administration of vectors showed a marked difference in transgene activity. Ad35-Luc gene expression peaked at 7 days and remained elevated for 6 months. Ad28-Luc expression was high after 1 day and remained sustained for one month.Different adenoviral vector serotypes ± modifications transduce different cells within the eye. Transgene expression can be brief or extended and is serotype and delivery route dependent. Thus, adenoviral vectors provide a versatile platform for the delivery of therapeutic agents for ocular diseases

Intraocular localization of GFP delivered by subretinal injections.

<p>Ad5-GFP injected eyes showed GFP fluorescence in the photoreceptors and RPE cells (A, B). Ad5+GFP injected eyes also showed GFP fluorescence in the photoreceptors and RPE cells (C, D). Ad35-GFP injection also showed GFP fluorescence in the photoreceptors and RPE cells (E, F). Ad35+GFP injection also showed GFP fluorescence in the photoreceptors and RPE cells (G, H). Ad28- transduced RPE cells alone (I, J). Ad28-Null injected eye demonstrated no obvious fluorescence (K, L). Ad5-GFP injected eyes demonstrated stronger GFP fluorescence in the RPE cells (A, B), and Ad5+GFP transduced photoreceptors dominantly (C, D). Scale bar = 50 µm.</p

Intraocular localization of GFP delivered by intravitreous injections.

<p>Ad5-GFP vectors transduced corneal endothelial cells, trabecular cells and iris cells (<b>A</b>). Ad5+GFP vectors transduced corneal endothelial cells, trabecular cells and iris cells (<b>B</b>). Ad35-GFP vectors transduced travecular cells alone (<b>C</b>). Ad35+GFP vectors transduced corneal endothelial cells, trabecular cells and iris cells (<b>D</b>). Ad28-GFP vectors transduced corneal endothelial cells, trabecular cells and iris cells (<b>E</b>). An Ad28-Null injected eye showed no GFP fluorescence in any ocular cells (<b>F</b>). Scale bar = 100 µm.</p

Summary of the localization and duration of transgene expression.

<p>Summary of the localization and duration of transgene expression.</p

Luciferase activity following single intravitreous injections of Ad5-Luc, Ad35-Luc and Ad28-Luc vectors.

<p>Luciferase activity peaked for all vectors within one day post-injection. Of all the tested vectors, the Ad5-Luc vector gave the greatest luciferase activity at Day 1. After Day 1, luciferase activity declined for all constructs and by Day 28 relative luciferase activity was very similar between the three vectors. Error bars: SEM. n = 6/vector/time point.</p

Luciferase activity following a single subretinal injection of Ad5-Luc, Ad35-Luc and Ad28-Luc.

<p>Ad5-Luc expression peaked one day after injection, but thereafter decreased quickly. Ad35-Luc expression peaked at seven days after injection and remained elevated out to 6 months. Ad35-Luc expression was significantly stronger than Ad5-Luc at the time point of 28 days and 6 months (<i>p</i> = 0.0127*, 0.033^‡, respectively). Ad28-Luc expression was substantial and was sustained throughout one month of observation, but was low at 6 months. Ad28-Luc expression was significantly stronger than Ad5-Luc at 28 days after injection (<i>p</i> = 0.0127^†). Error bars: SEM. n = 4 to 6/vector/time point. Which are Ad5-1day 4eyes, Ad35-1day 5 eyes, Ad28-1day 4 eyes, Ad5-7days 5 eyes, Ad35-7days 4 eyes, Ad28-7days 5 eyes, Ad5-14days 4 eyes, Ad35-14days 5 eyes, Ad28-14days 4 eyes, Ad5-28days 5 eyes, Ad35-28days 4 eyes, Ad28-28days 4 eyes, Ad5-6months 6 eyes, Ad35-6months 4 eyes, Ad28-6months 6 eyes.</p

Schematic diagram and abbreviation of Ad5, Ad35 and Ad28.

<p>Schematic diagram comparing similar regions in Ad5, Ad35 and Ad28 vectors (<b>A</b>). The E1 region has been deleted in each construct and replaced with the SV40 poly A region, the luciferase or green fluorescent protein cDNA, and the cytomegalovirus promoter (CMV). In the Ad5 vector, there is a partial E3 deletion and the E4 region was replaced with a transcriptionally inert sequence. In the Ad35 and Ad28 vectors, the E3 regions and E4 regions are intact. The genomes of all vectors are approximately the same size. <i>Plus</i> in the vector abbreviation means RGD motif incorporation in the fiber knob. Ad5+ alone has also been deleted for the RGD motif in the penton base (<b>B</b>).</p