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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 minimal cytomegalovirus (CMV) promoter showed increased Müller glial cell restricted expression via the intravitreal route. In vivo scanning laser ophthalmoscopy at 830 nm (left panel) for native fundus images and at 488 nm for green fluorescent protein (GFP) fluorescence (middle panel) of Crb1−/− mice 3 weeks after intravitreal injection of 109 genome copies (gc) of (a) AAV2/ShH10Y-minimal CMV-GFP showed GFP expression along and between the blood vessels, and a representative transverse retinal section (right panel) revealed many GFP-positive Müller glial cells. Confocal imaging of immunostaining of (b) glutamine synthetase (a marker for Müller glial cells), (c) protein kinase C α (PKCα; a marker for bipolar cells), (d) calretinin (a marker for amacrine cells), and (e) calbindin (a marker for horizontal and amacrine cells) showed GFP expression in Müller glial, amacrine, and ganglion cells and barely in horizontal cells. (f) Transduction profiles of three retinas injected with AAV2/ShH10Y full-length or minimal CMV-GFP at 109 gc revealed that the minimal CMV promoter mediated increased Müller glial cell restricted expression compared with the native CMV promoter. Data are presented as mean ± SEM and n = 3/promoter. *P < 0.05. Bar = 50 µm (a,d,e), 25 µm (b–c). AAV, adeno-associated virus; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PRC, photoreceptor cells; RPE, retinal pigment epithelium.
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fig7: The minimal cytomegalovirus (CMV) promoter showed increased Müller glial cell restricted expression via the intravitreal route. In vivo scanning laser ophthalmoscopy at 830 nm (left panel) for native fundus images and at 488 nm for green fluorescent protein (GFP) fluorescence (middle panel) of Crb1−/− mice 3 weeks after intravitreal injection of 109 genome copies (gc) of (a) AAV2/ShH10Y-minimal CMV-GFP showed GFP expression along and between the blood vessels, and a representative transverse retinal section (right panel) revealed many GFP-positive Müller glial cells. Confocal imaging of immunostaining of (b) glutamine synthetase (a marker for Müller glial cells), (c) protein kinase C α (PKCα; a marker for bipolar cells), (d) calretinin (a marker for amacrine cells), and (e) calbindin (a marker for horizontal and amacrine cells) showed GFP expression in Müller glial, amacrine, and ganglion cells and barely in horizontal cells. (f) Transduction profiles of three retinas injected with AAV2/ShH10Y full-length or minimal CMV-GFP at 109 gc revealed that the minimal CMV promoter mediated increased Müller glial cell restricted expression compared with the native CMV promoter. Data are presented as mean ± SEM and n = 3/promoter. *P < 0.05. Bar = 50 µm (a,d,e), 25 µm (b–c). AAV, adeno-associated virus; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PRC, photoreceptor cells; RPE, retinal pigment epithelium.

Mentions: A shorter and engineered version of the CMV promoter (0.26 kb) was designed with enhancer elements from the full-length CMV promoter.29,30 Three weeks after intravitreal injection of ShH10Y-minimal CMV-GFP, GFP was visible on SLO imaging along and between the blood vessels (Figure 7a). GFP-positive cells were mainly Müller glial cells (Figure 7b), with a few bipolar (Figure 7c) or amacrine cells (Figure 7d) and very few horizontal cells (Figure 7e). The minimal CMV promoter slightly modified the cell-specific expression profile, as more Müller glial cells were GFP positive (79 ± 3% for minimal CMV versus 64 ± 3% for full-length CMV; Figure 3f).


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 minimal cytomegalovirus (CMV) promoter showed increased Müller glial cell restricted expression via the intravitreal route. In vivo scanning laser ophthalmoscopy at 830 nm (left panel) for native fundus images and at 488 nm for green fluorescent protein (GFP) fluorescence (middle panel) of Crb1−/− mice 3 weeks after intravitreal injection of 109 genome copies (gc) of (a) AAV2/ShH10Y-minimal CMV-GFP showed GFP expression along and between the blood vessels, and a representative transverse retinal section (right panel) revealed many GFP-positive Müller glial cells. Confocal imaging of immunostaining of (b) glutamine synthetase (a marker for Müller glial cells), (c) protein kinase C α (PKCα; a marker for bipolar cells), (d) calretinin (a marker for amacrine cells), and (e) calbindin (a marker for horizontal and amacrine cells) showed GFP expression in Müller glial, amacrine, and ganglion cells and barely in horizontal cells. (f) Transduction profiles of three retinas injected with AAV2/ShH10Y full-length or minimal CMV-GFP at 109 gc revealed that the minimal CMV promoter mediated increased Müller glial cell restricted expression compared with the native CMV promoter. Data are presented as mean ± SEM and n = 3/promoter. *P < 0.05. Bar = 50 µm (a,d,e), 25 µm (b–c). AAV, adeno-associated virus; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PRC, photoreceptor cells; RPE, retinal pigment epithelium.
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fig7: The minimal cytomegalovirus (CMV) promoter showed increased Müller glial cell restricted expression via the intravitreal route. In vivo scanning laser ophthalmoscopy at 830 nm (left panel) for native fundus images and at 488 nm for green fluorescent protein (GFP) fluorescence (middle panel) of Crb1−/− mice 3 weeks after intravitreal injection of 109 genome copies (gc) of (a) AAV2/ShH10Y-minimal CMV-GFP showed GFP expression along and between the blood vessels, and a representative transverse retinal section (right panel) revealed many GFP-positive Müller glial cells. Confocal imaging of immunostaining of (b) glutamine synthetase (a marker for Müller glial cells), (c) protein kinase C α (PKCα; a marker for bipolar cells), (d) calretinin (a marker for amacrine cells), and (e) calbindin (a marker for horizontal and amacrine cells) showed GFP expression in Müller glial, amacrine, and ganglion cells and barely in horizontal cells. (f) Transduction profiles of three retinas injected with AAV2/ShH10Y full-length or minimal CMV-GFP at 109 gc revealed that the minimal CMV promoter mediated increased Müller glial cell restricted expression compared with the native CMV promoter. Data are presented as mean ± SEM and n = 3/promoter. *P < 0.05. Bar = 50 µm (a,d,e), 25 µm (b–c). AAV, adeno-associated virus; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PRC, photoreceptor cells; RPE, retinal pigment epithelium.
Mentions: A shorter and engineered version of the CMV promoter (0.26 kb) was designed with enhancer elements from the full-length CMV promoter.29,30 Three weeks after intravitreal injection of ShH10Y-minimal CMV-GFP, GFP was visible on SLO imaging along and between the blood vessels (Figure 7a). GFP-positive cells were mainly Müller glial cells (Figure 7b), with a few bipolar (Figure 7c) or amacrine cells (Figure 7d) and very few horizontal cells (Figure 7e). The minimal CMV promoter slightly modified the cell-specific expression profile, as more Müller glial cells were GFP positive (79 ± 3% for minimal CMV versus 64 ± 3% for full-length CMV; Figure 3f).

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