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Specific tools for targeting and expression in Müller glial cells.

Pellissier LP, Hoek RM, Vos RM, Aartsen WM, Klimczak RR, Hoyng SA, Flannery JG, Wijnholds J - Mol Ther Methods Clin Dev (2014)

Bottom Line: In this study, we characterized the efficiency of adeno-associated virus (AAV) capsid variants and different promoters to drive protein expression in Müller glial cells.We also developed and tested smaller promoters to express the large CRB1 gene via AAV vectors.Minimal cytomegalovirus (CMV) promoter allowed expression of full-length CRB1 protein in Müller 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
Despite their physiological roles, Müller glial cells are involved directly or indirectly in retinal disease pathogenesis and are an interesting target for therapeutic approaches for retinal diseases and regeneration such as CRB1 inherited retinal dystrophies. In this study, we characterized the efficiency of adeno-associated virus (AAV) capsid variants and different promoters to drive protein expression in Müller glial cells. ShH10Y and AAV9 were the most powerful capsids to infect mouse Müller glial cells. Retinaldehyde-binding protein 1 (RLBP1) promoter was the most powerful promoter to transduce Müller glial cells. ShH10Y capsids and RLBP1 promoter targeted human Müller glial cells in vitro. We also developed and tested smaller promoters to express the large CRB1 gene via AAV vectors. Minimal cytomegalovirus (CMV) promoter allowed expression of full-length CRB1 protein in Müller glial cells. In summary, ShH10Y and AAV9 capsids, and RLBP1 or minimal CMV promoters are of interest as specific tools to target and express in mouse or human Müller glial cells.

No MeSH data available.


Related in: MedlinePlus

The RLBP1 promoter drives strong green fluorescent protein (GFP) expression in Müller glial cells and retinal pigment epithelium (RPE) via the subretinal route. In vivo scanning laser ophthalmoscopy at 830 nm (left panel) for native fundus images and at 488 nm for GFP fluorescence (middle panel) of four Crb1−/− mice 3 weeks after subretinal injection of 109 genome copies of (a) AAV2/ShH10Y-RLBP1-GFP showed a typical pattern of GFP expression following subretinal injection, and a representative transverse retinal section (right panel) revealed that only Müller glial and RPE cells expressed GFP. Confocal imaging of GFP-positive cells coimmunostained with (b) glutamine synthetase, (c) SOX9, (d) native RLBP1 protein (Müller glial and RPE cells), (e) calretinin (a marker for amacrine cells), (f) cone arrestin (a marker for cones), and (g) recoverin (a marker for photoreceptors) showed only colocalization of GFP with glutamine synthetase, SOX9, and RLBP1. n = 4. Bar = 50 µm (a,b,e), 25 µm (c,d,f). AAV, adeno-associated virus; GCL, ganglion cell layer; INL, inner nuclear layer; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment; RLBP1, retinaldehyde binding protein 1; RPE, retinal pigment epithelium.
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fig5: The RLBP1 promoter drives strong green fluorescent protein (GFP) expression in Müller glial cells and retinal pigment epithelium (RPE) via the subretinal route. In vivo scanning laser ophthalmoscopy at 830 nm (left panel) for native fundus images and at 488 nm for GFP fluorescence (middle panel) of four Crb1−/− mice 3 weeks after subretinal injection of 109 genome copies of (a) AAV2/ShH10Y-RLBP1-GFP showed a typical pattern of GFP expression following subretinal injection, and a representative transverse retinal section (right panel) revealed that only Müller glial and RPE cells expressed GFP. Confocal imaging of GFP-positive cells coimmunostained with (b) glutamine synthetase, (c) SOX9, (d) native RLBP1 protein (Müller glial and RPE cells), (e) calretinin (a marker for amacrine cells), (f) cone arrestin (a marker for cones), and (g) recoverin (a marker for photoreceptors) showed only colocalization of GFP with glutamine synthetase, SOX9, and RLBP1. n = 4. Bar = 50 µm (a,b,e), 25 µm (c,d,f). AAV, adeno-associated virus; GCL, ganglion cell layer; INL, inner nuclear layer; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment; RLBP1, retinaldehyde binding protein 1; RPE, retinal pigment epithelium.

Mentions: When delivered subretinally, ShH10Y-RLBP1-GFP vectors showed on SLO a typical pattern of GFP expression (Figure 5a). Transverse sections revealed strong GFP expression restricted to RPE and Müller glial cells (Figure 5b–c). Endogenous staining for RLBP1 protein (RPE and Müller glial cells)28 showed strong colocalization with GFP (Figure 5d), whereas no colocalization was found with markers for other cell types (Figure 5e–g). In summary, when applied subretinally, the AAV9 RLBP1 vector drives expression in RPE and Müller glia cells, when delivered into the vitreous, the ShH10Y-RLBP1 promoter restricts expression to Müller glia cells.


Specific tools for targeting and expression in Müller glial cells.

Pellissier LP, Hoek RM, Vos RM, Aartsen WM, Klimczak RR, Hoyng SA, Flannery JG, Wijnholds J - Mol Ther Methods Clin Dev (2014)

The RLBP1 promoter drives strong green fluorescent protein (GFP) expression in Müller glial cells and retinal pigment epithelium (RPE) via the subretinal route. In vivo scanning laser ophthalmoscopy at 830 nm (left panel) for native fundus images and at 488 nm for GFP fluorescence (middle panel) of four Crb1−/− mice 3 weeks after subretinal injection of 109 genome copies of (a) AAV2/ShH10Y-RLBP1-GFP showed a typical pattern of GFP expression following subretinal injection, and a representative transverse retinal section (right panel) revealed that only Müller glial and RPE cells expressed GFP. Confocal imaging of GFP-positive cells coimmunostained with (b) glutamine synthetase, (c) SOX9, (d) native RLBP1 protein (Müller glial and RPE cells), (e) calretinin (a marker for amacrine cells), (f) cone arrestin (a marker for cones), and (g) recoverin (a marker for photoreceptors) showed only colocalization of GFP with glutamine synthetase, SOX9, and RLBP1. n = 4. Bar = 50 µm (a,b,e), 25 µm (c,d,f). AAV, adeno-associated virus; GCL, ganglion cell layer; INL, inner nuclear layer; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment; RLBP1, retinaldehyde binding protein 1; RPE, retinal pigment epithelium.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4362388&req=5

fig5: The RLBP1 promoter drives strong green fluorescent protein (GFP) expression in Müller glial cells and retinal pigment epithelium (RPE) via the subretinal route. In vivo scanning laser ophthalmoscopy at 830 nm (left panel) for native fundus images and at 488 nm for GFP fluorescence (middle panel) of four Crb1−/− mice 3 weeks after subretinal injection of 109 genome copies of (a) AAV2/ShH10Y-RLBP1-GFP showed a typical pattern of GFP expression following subretinal injection, and a representative transverse retinal section (right panel) revealed that only Müller glial and RPE cells expressed GFP. Confocal imaging of GFP-positive cells coimmunostained with (b) glutamine synthetase, (c) SOX9, (d) native RLBP1 protein (Müller glial and RPE cells), (e) calretinin (a marker for amacrine cells), (f) cone arrestin (a marker for cones), and (g) recoverin (a marker for photoreceptors) showed only colocalization of GFP with glutamine synthetase, SOX9, and RLBP1. n = 4. Bar = 50 µm (a,b,e), 25 µm (c,d,f). AAV, adeno-associated virus; GCL, ganglion cell layer; INL, inner nuclear layer; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment; RLBP1, retinaldehyde binding protein 1; RPE, retinal pigment epithelium.
Mentions: When delivered subretinally, ShH10Y-RLBP1-GFP vectors showed on SLO a typical pattern of GFP expression (Figure 5a). Transverse sections revealed strong GFP expression restricted to RPE and Müller glial cells (Figure 5b–c). Endogenous staining for RLBP1 protein (RPE and Müller glial cells)28 showed strong colocalization with GFP (Figure 5d), whereas no colocalization was found with markers for other cell types (Figure 5e–g). In summary, when applied subretinally, the AAV9 RLBP1 vector drives expression in RPE and Müller glia cells, when delivered into the vitreous, the ShH10Y-RLBP1 promoter restricts expression to Müller glia cells.

Bottom Line: In this study, we characterized the efficiency of adeno-associated virus (AAV) capsid variants and different promoters to drive protein expression in Müller glial cells.We also developed and tested smaller promoters to express the large CRB1 gene via AAV vectors.Minimal cytomegalovirus (CMV) promoter allowed expression of full-length CRB1 protein in Müller 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
Despite their physiological roles, Müller glial cells are involved directly or indirectly in retinal disease pathogenesis and are an interesting target for therapeutic approaches for retinal diseases and regeneration such as CRB1 inherited retinal dystrophies. In this study, we characterized the efficiency of adeno-associated virus (AAV) capsid variants and different promoters to drive protein expression in Müller glial cells. ShH10Y and AAV9 were the most powerful capsids to infect mouse Müller glial cells. Retinaldehyde-binding protein 1 (RLBP1) promoter was the most powerful promoter to transduce Müller glial cells. ShH10Y capsids and RLBP1 promoter targeted human Müller glial cells in vitro. We also developed and tested smaller promoters to express the large CRB1 gene via AAV vectors. Minimal cytomegalovirus (CMV) promoter allowed expression of full-length CRB1 protein in Müller glial cells. In summary, ShH10Y and AAV9 capsids, and RLBP1 or minimal CMV promoters are of interest as specific tools to target and express in mouse or human Müller glial cells.

No MeSH data available.


Related in: MedlinePlus