10 research outputs found
Recombinant Vectors Based on Porcine Adeno-Associated Viral Serotypes Transduce the Murine and Pig Retina
<div><p>Recombinant adeno-associated viral (AAV) vectors are known to safely and efficiently transduce the retina. Among the various AAV serotypes available, AAV2/5 and 2/8 are the most effective for gene transfer to photoreceptors (PR), which are the most relevant targets for gene therapy of inherited retinal degenerations. However, the search for novel AAV serotypes with improved PR transduction is ongoing. In this work we tested vectors derived from five AAV serotypes isolated from porcine tissues (referred to as porcine AAVs, four of which are newly identified) for their ability to transduce both the murine and the cone-enriched pig retina. Porcine AAV vectors expressing EGFP under the control of the CMV promoter were injected subretinally either in C57BL/6 mice or Large White pigs. The resulting retinal tropism was analyzed one month later on histological sections, while levels of PR transduction were assessed by Western blot. Our results show that all porcine AAV transduce murine and porcine retinal pigment epithelium and PR upon subretinal administration. AAV2/po1 and 2/po5 are the most efficient porcine AAVs for murine PR transduction and exhibit the strongest tropism for pig cone PR. The levels of PR transduction obtained with AAV2/po1 and 2/po5 are similar, albeit not superior, to those obtained with AAV2/5 and AAV2/8, which evinces AAV2/po1 and 2/po5 to be promising vectors for retinal gene therapy.</p> </div
Phylogenetic tree obtained through the alignment of the <i>cap</i> sequences of the newly isolated porcine serotypes and other preexisting AAVs.
<p>Phylogenetic tree obtained through the alignment of the <i>cap</i> sequences of the newly isolated porcine serotypes and other preexisting AAVs.</p
Quantification of PR-specific EGFP expression following subretinal delivery of AAV vectors.
<p><b>A:</b> Representative Western blot out of five, each containing lysates (50 μg/lane) from retinas injected subretinally one month before with the various AAV vectors (indicated above each lane) containing the Rho promoter (6E9 GC/eye). Each blot was incubated with anti-EGFP (upper panel, α-EGFP) and anti-β-tubulin (lower panel, for normalization, α-β-tubulin) antibodies. The molecular weight is indicated on the left. <b>B:</b> Quantification of PR-specific EGFP expression mediated by each AAV vector (indicated below each bar), compared to AAV2/5. The intensity of the EGFP and β-tubulin bands was quantified and the resulting EGFP/β-tubulin ratio values were divided by those of AAV2/5. Averages (indicated above or inside each bar) ± SE for the 5 retinas injected with each AAV have been plotted. Statistical differences were calculated using one-way ANOVA (p-value  = 1,87E-5). * corresponds to p≤0.05, ** to p≤0.01, *** to p≤0.001 and **** to p≤0.0001.</p
Murine retinal transduction following AAV subretinal delivery.
<p>AAV vectors (2E9 GC/eye with the exception of AAV/po1 which was injected at 5E9 GC/eye) were delivered subretinally in adult C57Bl/6 mice. One month later non-invasive fundus photographs (upper panels; photograph not available for AAV2/po4) were taken to monitor fluorescence before sacrifice, when fluorescence microscope analysis of histological sections (lower panels) was performed. n indicates the number of retinal sections resembling the representative picture out of the total number of injected retinas. The AAV vector serotypes used are indicated above each panel. RPE: retinal pigment epithelium; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. Magnification of histological sections  = 20×; scale bar  = 20 µm; exposure  = 1 sec.</p
Subretinal delivery of AAV vectors does not alter porcine retinal histology.
<p>Hematoxylin and eosin staining of pig retina cryosections one month after transduction with 1E10 GC/eye of each AAV vector (indicated above each picture), as in Fig. 2. Non-injected retinas were used as controls. RPE: retinal pigment epithelium; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. Magnification  = 20×; scale bar  = 50 µm.</p
Amino acid (aa) sequence alignment of AAV5, −8, -po1, -po2.1, -po4, -po5, and -po6.
<p>Dots indicate conserved residues with AAV8, while dashes represent aa missing at those positions. The clear boxes are putative sialic acid binding domains of AAV5.</p
Ganzfeld electroretinogram (ERG) maximum amplitude values in retinas non-injected or injected with AAV2/5, 2/8 and porcine AAV vectors.
<p>NB: Values in the table are the average maximum a-waves and b-waves amplitudes obtained by ERG under scotopic and photopic conditions. No statistically significant differences were found between values of non-injected (n = 3) retinas and injected with AAV vectors (n = 5 for each serotype). SE: standard error.</p
Murine PR transduction mediated by AAV vectors containing the rod-specific Rho promoter.
<p>Rho-driven EGFP fluorescence in murine retinal sections one month after subretinal delivery of AAV vectors (6E9 GC/eye). The AAV serotypes used and the n of retinas injected are indicated above each column. Magnification  = 10×; exposure  = 6 s.</p
Pig cone transduction following subretinal administration of AAV vectors.
<p><b>A:</b> Pig retinal cryosections were immunolabelled with the cone-specific anti-cone arrestin (CAR) antibody one month after subretinal delivery of AAV vectors (indicated above each picture), as in Fig. 2. Double-labelled transduced cones (EGFP+/CAR+) are shown in the insets. Confocal microscope magnification  = 63×; scale bar  = 50 µm. <b>B:</b> Quantification of cone transduction mediated by porcine AAV serotypes. The number of EGFP+/CAR+ cells obtained with each serotype was divided by the number of EGFP+/CAR+ cells obtained with AAV2/5. Values are shown as average (indicated inside each bar) ± SE. Statistical differences were calculated using GLM (p-value  = 8.6E-7). ** corresponds to p≤0.01, *** to p≤0.001 and **** to p≤0.0001.</p
Porcine retinal transduction following AAV subretinal delivery.
<p>AAV vectors were delivered subretinally in pigs at a dose of 1E10 GC/eye. One month later eyes were harvested and retinal histological sections were analyzed by fluorescence microscopy. The n of retinas analyzed is indicated at the bottom of each panel. The AAV vector serotypes used are indicated above each panel. RPE: retinal pigment epithelium; ONL: outer nuclear layer; INL: inner nuclear layer. Magnification  = 20×; scale bar  = 50 µm; exposure  = 2 sec.</p