Real-Time PCR analysis of vector genome (a,b,c) and mRNA (d,e,f) levels following AAV2/8 transduction of mouse cornea.

<p>Amplification curves of (<b>a</b>) GAPDH and (<b>b</b>) EGFP in DNA extracted from injected mouse eyes. To aid visualisation, only one of the duplicate curves is shown. (<b>c</b>) Graphical representation of the concentration of EGFP normalised to that of GAPDH in each sample (the colour of the curves in <b>a and b</b> matches those of the corresponding bars in <b>c</b>). As a control, EGFP DNA could not be detected following injection and re-injection of the mouse cornea with PBS (PBS PBS). In contrast, EGFP DNA levels were detected and relatively stable both 8- and 14-d post-AAV2/8 transduction (blue bars). Following a second injection of PBS 7-d post-transduction, a 1.9- and 4-fold decrease in EGFP DNA was detected on days 8 and 14, respectively (red bars). Amplification curves of (<b>d</b>) GAPDH and (<b>e</b>) EGFP in RNA extracted from injected mouse eyes. (<b>f</b>) Graphical representation of the concentration of EGFP normalised to that of GAPDH in each sample (the colour of the curves in <b>d and e</b> matches those of the corresponding bars in <b>f</b>). As a control, EGFP RNA levels could not be detected following injection and re-injection of the mouse cornea with PBS. Similarly, EGFP RNA levels could not be detected 8- and 14-d post AAV2/8 transduction. In contrast, a second injection of PBS 7-d post-transduction resulted in a 65-fold increase in EGFP RNA levels on day 8 (red bar). RNA levels were no longer detectable by day 14.</p

Transduction efficiency of the AAV vectors in the human corneal explants.

<p>One-wk post-injection, using <i>in vivo</i> microscopy, EGFP expression can be seen in throughout the cornea following intra-stromal injection of the vectors AAV2/1 (<b>a</b>) and AAV2/8 (<b>i</b>). (<b>b</b>, <b>j</b>) Higher magnification of the boxed regions in <b>a</b> and <b>i</b>, respectively. (<b>e</b>) Three-weeks post transduction, EGFP expression can be detected following AAV2/2 injection. (<b>f</b>) Higher magnification of the boxed area in <b>e</b>. (<b>c</b>, <b>g</b>, <b>k</b>) EGFP expression on histological sections of each cornea 8-wk post-injection of AAV2/1, −/2, −/8, respectively. (<b>d</b>, <b>h</b>, <b>l</b>) Imaris-treated images of <b>c</b>, <b>g</b>, <b>k</b>, respectively, showing EGFP-expressing cells.</p

Transduction efficiency of AAV vectors in the mouse cornea.

<p>EGFP expression (indicated by arrows) in the mouse cornea detected by <i>in vivo</i> epifluorescence microscopy 1-wk post-injection of AAV2/1 (<b>a</b>), AAV2/2 (<b>e</b>), AAV2/5 (<b>i</b>) and AAV2/8 (<b>m</b>) vectors. (<b>b</b>, <b>f</b>, <b>j</b>, <b>n</b>) Higher magnification of <b>a</b>, <b>e</b>, <b>i</b> and <b>m</b>, respectively. (<b>c</b>, <b>g</b>, <b>k</b>, <b>o</b>) EGFP expression in the same corneas detected 4-wk post-injection. (<b>d</b>, <b>h</b>, <b>l</b>, <b>p</b>) Higher magnification of <b>c</b>, <b>g</b>, <b>k</b> and <b>o</b>, respectively. Magnifications <b>a</b>, <b>e</b>, <b>g</b>, <b>i</b>, <b>k</b>, <b>m</b>, <b>o</b>: 20×; <b>b</b>: 43×; <b>c</b>: 25×, <b>d</b>: 53×; <b>f</b>, <b>j</b>: 35×; <b>h</b>: 44×, <b>l</b>: 33×; <b>n</b>: 40×; <b>p</b>: 45×. For reference, the diameter of an adult mouse eye is ∼3.5 mm. The large green spot in centre of photos is the pupil of the mouse eye. The asterisk in panels <b>i</b> to <b>l</b> indicates an opaque lesion on the mouse eye that was present from the beginning of the experiments.</p

Intra-stromal injection of AAV2/8 in the human cornea results in transduction of keratocytes.

<p>(<b>a–d</b>) Anti-CD34 labelling (in red) of a human cornea co-localises with EGFP-expressing cells (in green) and identifies these cells as quiescent keratocytes. (<b>e–h</b>) Anti-α–SMA labelling (in red) of a human cornea co-localises with EGFP-expressing cells (in green) and identifies these cells as activated keratocytes.</p

EGFP-expressing cell population following PBS injection in the transduced mouse cornea.

<p>(<b>a</b>–<b>d</b>) Anti-CD34 staining (in red) of a mouse cornea 24 h post-PBS injection (same eye shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035318#pone-0035318-g004" target="_blank">Fig. 4C</a>). The CD34 signal co-localises with the EGFP signal (arrows and arrowheads). (<b>e</b>–<b>f</b>) Anti-F4/80 staining (in red) of a mouse cornea 24 h post-PBS injection. The F4/80 signal does not co-localise with the EGFP signal (arrows).</p

Schematic representation of the events following PBS injection and hyper-activation of EGFP expression.

<p>(<b>a</b>) Following initial AAV2/8 injection, a large number of cells harbour viral particles or vector genomes but a small number of cells express EGFP (in green). (<b>b</b>) Twenty-four h post-PBS injection, the disruption of the epithelial basement membrane results in the release of cytokines that induce the repair process. This process involves cell death in the stroma immediately underlying the injection site, which reduces the vector genome levels (∼2-fold decrease), and cell migration from the limbal region. The ensuing events also result in either <i>de novo</i> uncoating of encapsidated virions or reactivation of transcriptionally-silenced genomes leading to activated EGFP expression (consistent with the 65-fold increase in EGFP mRNA levels). (<b>c</b>) One-week post-PBS injection, the cornea has returned to its basal state and is no longer expressing EGFP (low mRNA levels). The cell death process has removed a number of transduced keratocytes (4-fold decrease in vector genome levels). However, cells harbouring the virions are still present (persisting DNA levels) as a subsequent PBS injection still can activate EGFP expression but less extensively.</p

Cell specificity of AAV2/8 transduction.

<p>(<b>a</b>–<b>d</b>) Anti-CD34 staining (in red) of a non-injected mouse cornea shows abundant CD34+ cells. (<b>e</b>–<b>h</b>) Anti-CD34 labelling (in red) of a mouse cornea 24 h post-injection with 3×10⁹ vg of AAV2/8. Intra-stromal injection results in a decrease in the proportion of CD34+ cells (in red) due to an augmentation in the number of cell nuclei (in blue). The EGFP signal co-localises (arrows and arrowheads) with the CD34-labelled cells. Inset in <b>h</b> shows a higher magnification of the cells indicated by arrowheads minus the Hoechst filter. (<b>i</b>–<b>l</b>) Anti-F4/80 staining (in red) of a non-injected mouse cornea showing a low number of F4/80+ cells. (<b>m</b>–<b>p</b>) Anti-F4/80 labelling (in red) of a mouse cornea 24 h post-injection with 3×10⁹ vg of AAV2/8. The EGFP signal does not co-localise (arrows) with the F4/80-labelled cells.</p

Molecular strategy followed up for the diagnosis of CHM families.

<p>Molecular strategy followed up for the diagnosis of CHM families.</p

Mutational spectrum of CHM characterized families.

<p>Mutational spectrum of CHM characterized families.</p

Haplotypes from families presenting the recurrent <i>CHM</i> mutations.

<p>Identified pedigrees carrying the exon 9 deletion <b>(A)</b>, the p.Arg293* <b>(B)</b> and the p.Lys178Argfs*5 <b>(C)</b> mutations are shown. For exon 9 deletion, haplotypes analysis demonstrated identity by descent in the Spanish families RP-1310, RP-1560 and RP-2128 but independent origin for the Portuguese family RP-0779, defined by the alleles located along the black bar. For the p.Arg293* and p.Lys178Argfs*5 mutations, haplotypes indicates an independent origin for both variants defined by the alleles located along the black bar.</p