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GFAP-driven GFP expression in activated mouse Müller glial cells aligning retinal blood vessels following intravitreal injection of AAV2/6 vectors.

Aartsen WM, van Cleef KW, Pellissier LP, Hoek RM, Vos RM, Blits B, Ehlert EM, Balaggan KS, Ali RR, Verhaagen J, Wijnholds J - PLoS ONE (2010)

Bottom Line: We used adeno-associated viral AAV2/6 vectors to transduce mouse retinas.Light exposure of Crb1(-/-) retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Müller glial cells aligning retinal blood vessels.Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuromedical Genetics, Netherlands Institute for Neuroscience, an Institute of the Royal Netherlands Academy of Arts and Sciences, Amsterdam, The Netherlands.

ABSTRACT

Background: Müller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Müller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy.

Methodology/principal findings: We used adeno-associated viral AAV2/6 vectors to transduce mouse retinas. The transduction pattern of AAV2/6 was investigated by studying expression of the green fluorescent protein (GFP) transgene using scanning-laser ophthalmoscopy and immuno-histochemistry. AAV2/6 vectors transduced mouse Müller glial cells aligning the retinal blood vessels. However, the transduction capacity was hindered by the inner limiting membrane (ILM) and besides Müller glial cells, several other inner retinal cell types were transduced. To obtain Müller glial cell-specific transgene expression, the cytomegalovirus (CMV) promoter was replaced by the glial fibrillary acidic protein (GFAP) promoter. Specificity and activation of the GFAP promoter was tested in a mouse model for retinal gliosis. Mice deficient for Crumbs homologue 1 (CRB1) develop gliosis after light exposure. Light exposure of Crb1(-/-) retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Müller glial cells aligning retinal blood vessels.

Conclusions/significance: Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells.

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Flow cytometric analysis of AAV2/6-CMV-GFP vs AAV2/6-CAG-GFP transduced retinas.Representative example of 7-AAD staining; the negative (live) population is used for further analysis (A). A representative comparison of retinal cells from eyes transduced via intravitreal injection of either AAV2/6-CMV-GFP (blue) or AAV2/6-CAG-GFP (red) resulted in a population of GFP-positive cells. The left edge of the gate for GFP-positive cells was set using non-transduced retinal cells (gray) (B). The inset shows the mean (+SEM) of the fraction of the live cells that are GFP-positive in CAG-GFP compared to CMV-GFP eyes (n = 5). These measurements show no statistically significant differences (p>0.6, Mann-Whitney test).
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pone-0012387-g003: Flow cytometric analysis of AAV2/6-CMV-GFP vs AAV2/6-CAG-GFP transduced retinas.Representative example of 7-AAD staining; the negative (live) population is used for further analysis (A). A representative comparison of retinal cells from eyes transduced via intravitreal injection of either AAV2/6-CMV-GFP (blue) or AAV2/6-CAG-GFP (red) resulted in a population of GFP-positive cells. The left edge of the gate for GFP-positive cells was set using non-transduced retinal cells (gray) (B). The inset shows the mean (+SEM) of the fraction of the live cells that are GFP-positive in CAG-GFP compared to CMV-GFP eyes (n = 5). These measurements show no statistically significant differences (p>0.6, Mann-Whitney test).

Mentions: Histological analysis was performed to identify the transduced cell types. Localization of GFP after intravitreal injection of AAV2/6-CMV-GFP at three weeks of age is shown in Figure 2A and revealed the transduction of ganglion cells (asterisk), Müller glial cells (arrow), horizontal, amacrine and bipolar cells (not indicated). Transduced Müller glial cells were easily recognized by morphological characteristics, as these cells extend from the ILM to the OLM of the retina having their cell bodies in the inner nuclear layer (INL). To further corroborate transduction of Müller glial cells (arrow), the sections were stained for the Müller glial cell-specific marker; glutamine synthetase (Fig. 2B). Following intravitreal injection of AAV2/6-CMV-GFP at P1, mainly ganglion cells (asterisk) and sporadically photoreceptor cells (arrow head) close to the optic nerve were found positive for GFP (Fig. 2C). SLO images at three weeks of age showed that transduced cells were mainly present in the proximity of larger blood vessels. The relationship between the blood vessels and transduced cells is depicted in Figure 2D, showing a blood vessel surrounded by transduced Müller glial cells. In contrast, analysis of sections from eyes that had received subretinal injections at three weeks of age revealed that mainly RPE cells were transduced (Fig. 2E). Other transduced cell types, such as photoreceptor cells (arrow heads), could be seen only near the site of injection. Substitution of the CMV for the CAG promoter did not result in an altered transduction pattern (Fig. 2F). Flow cytometric analysis revealed a relatively high variability in the number of transduced cells within the examined retinas, but neither the GFP expression levels nor the number of transduced cells showed statistically significant difference when comparing the CMV promoter with the CAG promoter, using the Mann-Whitney test (Fig. 3A and B).


GFAP-driven GFP expression in activated mouse Müller glial cells aligning retinal blood vessels following intravitreal injection of AAV2/6 vectors.

Aartsen WM, van Cleef KW, Pellissier LP, Hoek RM, Vos RM, Blits B, Ehlert EM, Balaggan KS, Ali RR, Verhaagen J, Wijnholds J - PLoS ONE (2010)

Flow cytometric analysis of AAV2/6-CMV-GFP vs AAV2/6-CAG-GFP transduced retinas.Representative example of 7-AAD staining; the negative (live) population is used for further analysis (A). A representative comparison of retinal cells from eyes transduced via intravitreal injection of either AAV2/6-CMV-GFP (blue) or AAV2/6-CAG-GFP (red) resulted in a population of GFP-positive cells. The left edge of the gate for GFP-positive cells was set using non-transduced retinal cells (gray) (B). The inset shows the mean (+SEM) of the fraction of the live cells that are GFP-positive in CAG-GFP compared to CMV-GFP eyes (n = 5). These measurements show no statistically significant differences (p>0.6, Mann-Whitney test).
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Related In: Results  -  Collection

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pone-0012387-g003: Flow cytometric analysis of AAV2/6-CMV-GFP vs AAV2/6-CAG-GFP transduced retinas.Representative example of 7-AAD staining; the negative (live) population is used for further analysis (A). A representative comparison of retinal cells from eyes transduced via intravitreal injection of either AAV2/6-CMV-GFP (blue) or AAV2/6-CAG-GFP (red) resulted in a population of GFP-positive cells. The left edge of the gate for GFP-positive cells was set using non-transduced retinal cells (gray) (B). The inset shows the mean (+SEM) of the fraction of the live cells that are GFP-positive in CAG-GFP compared to CMV-GFP eyes (n = 5). These measurements show no statistically significant differences (p>0.6, Mann-Whitney test).
Mentions: Histological analysis was performed to identify the transduced cell types. Localization of GFP after intravitreal injection of AAV2/6-CMV-GFP at three weeks of age is shown in Figure 2A and revealed the transduction of ganglion cells (asterisk), Müller glial cells (arrow), horizontal, amacrine and bipolar cells (not indicated). Transduced Müller glial cells were easily recognized by morphological characteristics, as these cells extend from the ILM to the OLM of the retina having their cell bodies in the inner nuclear layer (INL). To further corroborate transduction of Müller glial cells (arrow), the sections were stained for the Müller glial cell-specific marker; glutamine synthetase (Fig. 2B). Following intravitreal injection of AAV2/6-CMV-GFP at P1, mainly ganglion cells (asterisk) and sporadically photoreceptor cells (arrow head) close to the optic nerve were found positive for GFP (Fig. 2C). SLO images at three weeks of age showed that transduced cells were mainly present in the proximity of larger blood vessels. The relationship between the blood vessels and transduced cells is depicted in Figure 2D, showing a blood vessel surrounded by transduced Müller glial cells. In contrast, analysis of sections from eyes that had received subretinal injections at three weeks of age revealed that mainly RPE cells were transduced (Fig. 2E). Other transduced cell types, such as photoreceptor cells (arrow heads), could be seen only near the site of injection. Substitution of the CMV for the CAG promoter did not result in an altered transduction pattern (Fig. 2F). Flow cytometric analysis revealed a relatively high variability in the number of transduced cells within the examined retinas, but neither the GFP expression levels nor the number of transduced cells showed statistically significant difference when comparing the CMV promoter with the CAG promoter, using the Mann-Whitney test (Fig. 3A and B).

Bottom Line: We used adeno-associated viral AAV2/6 vectors to transduce mouse retinas.Light exposure of Crb1(-/-) retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Müller glial cells aligning retinal blood vessels.Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuromedical Genetics, Netherlands Institute for Neuroscience, an Institute of the Royal Netherlands Academy of Arts and Sciences, Amsterdam, The Netherlands.

ABSTRACT

Background: Müller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Müller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy.

Methodology/principal findings: We used adeno-associated viral AAV2/6 vectors to transduce mouse retinas. The transduction pattern of AAV2/6 was investigated by studying expression of the green fluorescent protein (GFP) transgene using scanning-laser ophthalmoscopy and immuno-histochemistry. AAV2/6 vectors transduced mouse Müller glial cells aligning the retinal blood vessels. However, the transduction capacity was hindered by the inner limiting membrane (ILM) and besides Müller glial cells, several other inner retinal cell types were transduced. To obtain Müller glial cell-specific transgene expression, the cytomegalovirus (CMV) promoter was replaced by the glial fibrillary acidic protein (GFAP) promoter. Specificity and activation of the GFAP promoter was tested in a mouse model for retinal gliosis. Mice deficient for Crumbs homologue 1 (CRB1) develop gliosis after light exposure. Light exposure of Crb1(-/-) retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Müller glial cells aligning retinal blood vessels.

Conclusions/significance: Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells.

Show MeSH
Related in: MedlinePlus