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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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Related in: MedlinePlus

Transduction profiles of AAV2/6-CMV-EGFP retinas with or without collagenase treatement.Representative retinal slices from injected eyes were quantified for the number of each transduced cell type. Specific markers were used to determine the different cell types and to generate histograms comparing tropism profiles with (B) and without collagenase treatment (A). Transduction efficiencies were calculated based on the ratio of each cell type infected relative to the total number of EGFP-positive cells (n = 9). Error bars represent standard deviation among sample population.
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pone-0012387-g005: Transduction profiles of AAV2/6-CMV-EGFP retinas with or without collagenase treatement.Representative retinal slices from injected eyes were quantified for the number of each transduced cell type. Specific markers were used to determine the different cell types and to generate histograms comparing tropism profiles with (B) and without collagenase treatment (A). Transduction efficiencies were calculated based on the ratio of each cell type infected relative to the total number of EGFP-positive cells (n = 9). Error bars represent standard deviation among sample population.

Mentions: The ILM is thinner at the site of the major retinal blood vessels [25] and is known to be a major obstacle to cell migration after stem cell transplantation from the vitreal side of the retina [26]. This phenomenon might explain the typical transduction pattern along the major blood vessels observed following intravitreal administration of AAV2/6 viral particles. To overcome this barrier, AAV2/6-CMV-GFP was intravitreally administrated after disruption of the ILM by collagenase VII treatment [27]. The integrity of the retinas was studied by SLO (Fig. 4A), which revealed acute retinal hemorrhage in 2 out of 4 wild-type and all Crb1−/− retinas (data not shown). While the left eye of both wild-type and Crb1−/− mice was left untreated and showed the same transduction pattern as depicted in Figure 1B, the collagenase-treated right eye was much more efficiently transduced (Fig. 4B). Increased transduction was observed in all wild-type and Crb1−/− retinas tested (n = 4 and n = 5, respectively). Immunohistochemical analysis showed GFP expression throughout the retina, with expression in many retinal cell types of the inner retina but not in photoreceptor cells (Fig. 4C). We confirmed the transduction of Müller glial cells by co-staining with antibodies against glutamine synthetase (Fig. 4D). Transduction profiles were established using cell type specific markers (for horizontal cells we used Prox1, and for ganglion and amacrine cells calretinin) comparing transduced retinas with and without collagenase treatment (Fig. 5A and B). No significant changes in the transduction profile were found after collagenase treatment.


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)

Transduction profiles of AAV2/6-CMV-EGFP retinas with or without collagenase treatement.Representative retinal slices from injected eyes were quantified for the number of each transduced cell type. Specific markers were used to determine the different cell types and to generate histograms comparing tropism profiles with (B) and without collagenase treatment (A). Transduction efficiencies were calculated based on the ratio of each cell type infected relative to the total number of EGFP-positive cells (n = 9). Error bars represent standard deviation among sample population.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2927518&req=5

pone-0012387-g005: Transduction profiles of AAV2/6-CMV-EGFP retinas with or without collagenase treatement.Representative retinal slices from injected eyes were quantified for the number of each transduced cell type. Specific markers were used to determine the different cell types and to generate histograms comparing tropism profiles with (B) and without collagenase treatment (A). Transduction efficiencies were calculated based on the ratio of each cell type infected relative to the total number of EGFP-positive cells (n = 9). Error bars represent standard deviation among sample population.
Mentions: The ILM is thinner at the site of the major retinal blood vessels [25] and is known to be a major obstacle to cell migration after stem cell transplantation from the vitreal side of the retina [26]. This phenomenon might explain the typical transduction pattern along the major blood vessels observed following intravitreal administration of AAV2/6 viral particles. To overcome this barrier, AAV2/6-CMV-GFP was intravitreally administrated after disruption of the ILM by collagenase VII treatment [27]. The integrity of the retinas was studied by SLO (Fig. 4A), which revealed acute retinal hemorrhage in 2 out of 4 wild-type and all Crb1−/− retinas (data not shown). While the left eye of both wild-type and Crb1−/− mice was left untreated and showed the same transduction pattern as depicted in Figure 1B, the collagenase-treated right eye was much more efficiently transduced (Fig. 4B). Increased transduction was observed in all wild-type and Crb1−/− retinas tested (n = 4 and n = 5, respectively). Immunohistochemical analysis showed GFP expression throughout the retina, with expression in many retinal cell types of the inner retina but not in photoreceptor cells (Fig. 4C). We confirmed the transduction of Müller glial cells by co-staining with antibodies against glutamine synthetase (Fig. 4D). Transduction profiles were established using cell type specific markers (for horizontal cells we used Prox1, and for ganglion and amacrine cells calretinin) comparing transduced retinas with and without collagenase treatment (Fig. 5A and B). No significant changes in the transduction profile were found after collagenase treatment.

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