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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

AAV6, ShH10, and ShH10Y tropism following intravitreal injection in adult murine retina. In vivo scanning laser ophthalmoscopy at 830 nm (left panel) for native fundus images and at 488 nm for green fluorescent protein (GFP) fluorescence images (middle panel) of Crb1−/− mice 3 weeks after intravitreal injection of 1010 genome copies (gc) of CMV-GFP with (a) AAV6 capsids and AAV6-derived capsids (b) ShH10 and (c) ShH10Y showed more widespread expression of GFP with ShH10 and ShH10Y capsids than AAV6, which localized mainly along the blood vessels. A representative transverse retinal section (right panel) revealed more GFP-positive Müller glial cells with ShH10Y capsids. Transduction profiles of three retinas injected with CMV-GFP vectors packaged in AAV6, ShH10, and ShH10Y capsids at (d) 108, (e) 109, and (f) 1010 gc showed that ShH10Y is the most powerful capsid to transduce mouse Müller glial cells even at low doses. Data are presented as mean ± SEM and n = 3/AAV/dose. **P < 0.01, ***P < 0.001. Bar = 50 µm (a–c). AAV, adeno-associated virus; CMV, cytomegalovirus; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PRC, photoreceptor cell; RPE, retinal pigment epithelium.
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fig1: AAV6, ShH10, and ShH10Y tropism following intravitreal injection in adult murine retina. In vivo scanning laser ophthalmoscopy at 830 nm (left panel) for native fundus images and at 488 nm for green fluorescent protein (GFP) fluorescence images (middle panel) of Crb1−/− mice 3 weeks after intravitreal injection of 1010 genome copies (gc) of CMV-GFP with (a) AAV6 capsids and AAV6-derived capsids (b) ShH10 and (c) ShH10Y showed more widespread expression of GFP with ShH10 and ShH10Y capsids than AAV6, which localized mainly along the blood vessels. A representative transverse retinal section (right panel) revealed more GFP-positive Müller glial cells with ShH10Y capsids. Transduction profiles of three retinas injected with CMV-GFP vectors packaged in AAV6, ShH10, and ShH10Y capsids at (d) 108, (e) 109, and (f) 1010 gc showed that ShH10Y is the most powerful capsid to transduce mouse Müller glial cells even at low doses. Data are presented as mean ± SEM and n = 3/AAV/dose. **P < 0.01, ***P < 0.001. Bar = 50 µm (a–c). AAV, adeno-associated virus; CMV, cytomegalovirus; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PRC, photoreceptor cell; RPE, retinal pigment epithelium.

Mentions: We studied the transduction and expression profiles of AAV6, ShH10, and ShH10Y, (ShH10Y carries an additional tyrosine to phenylalanine mutation to enhance its transduction efficiency).21 We intravitreally injected Crb1−/− retinas with 108, 109, 1010 genome copies (gc) of CMV-GFP transgene packaged in the different AAV serotypes. We analyzed the GFP expression by scanning laser ophthalmoscopy (SLO) and immunohistochemistry using cell type–specific immunomarkers in retinal sections. At 108 gc, the ShH10Y variant showed enhanced ability to transduce Müller glial cells relative to the unmodified ShH10 and AAV6 capsids (49 ± 6, 24 ± 3, and 21 ± 3%, respectively; Figure 1d), the total number of GFP-positive cells per millimeter was not different (28 ± 4, 29 ± 6, and 26 ± 3%, respectively). At one log unit higher titer (109 gc), ShH10Y also showed the highest percentage of transduced mouse Müller glial cells, whereas ShH10 and AAV6 transduced less efficiently (66 ± 4, 23 ± 2, and 17 ± 6%, respectively; Figure 1e), the total number of infected cells per millimeter was not different (49 ± 2, 47 ± 10, and 51 ± 2, respectively). At 1010 gc, the cell types infected by AAV6 were primarily around the blood vessels as shown previously,14 whereas both ShH10 and ShH10Y showed a broader transduction pattern (Figure 1a–c). 62 ± 2% of the cells transduced by ShH10Y were Müller glial cells, whereas significantly less Müller glial cells were transduced with ShH10 (39 ± 1%) or AAV6 (18 ± 1%) (Figure 1a–c,f). The number of GFP-positive cells per millimeter was not significantly different (152 ± 25, 123 ± 44, and 147 ± 17, respectively). In summary, intravitreally injected ShH10Y vectors showed higher transduction efficiency for mouse Müller glial cells than AAV6 or ShH10 vectors.


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)

AAV6, ShH10, and ShH10Y tropism following intravitreal injection in adult murine retina. In vivo scanning laser ophthalmoscopy at 830 nm (left panel) for native fundus images and at 488 nm for green fluorescent protein (GFP) fluorescence images (middle panel) of Crb1−/− mice 3 weeks after intravitreal injection of 1010 genome copies (gc) of CMV-GFP with (a) AAV6 capsids and AAV6-derived capsids (b) ShH10 and (c) ShH10Y showed more widespread expression of GFP with ShH10 and ShH10Y capsids than AAV6, which localized mainly along the blood vessels. A representative transverse retinal section (right panel) revealed more GFP-positive Müller glial cells with ShH10Y capsids. Transduction profiles of three retinas injected with CMV-GFP vectors packaged in AAV6, ShH10, and ShH10Y capsids at (d) 108, (e) 109, and (f) 1010 gc showed that ShH10Y is the most powerful capsid to transduce mouse Müller glial cells even at low doses. Data are presented as mean ± SEM and n = 3/AAV/dose. **P < 0.01, ***P < 0.001. Bar = 50 µm (a–c). AAV, adeno-associated virus; CMV, cytomegalovirus; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PRC, photoreceptor cell; RPE, retinal pigment epithelium.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4362388&req=5

fig1: AAV6, ShH10, and ShH10Y tropism following intravitreal injection in adult murine retina. In vivo scanning laser ophthalmoscopy at 830 nm (left panel) for native fundus images and at 488 nm for green fluorescent protein (GFP) fluorescence images (middle panel) of Crb1−/− mice 3 weeks after intravitreal injection of 1010 genome copies (gc) of CMV-GFP with (a) AAV6 capsids and AAV6-derived capsids (b) ShH10 and (c) ShH10Y showed more widespread expression of GFP with ShH10 and ShH10Y capsids than AAV6, which localized mainly along the blood vessels. A representative transverse retinal section (right panel) revealed more GFP-positive Müller glial cells with ShH10Y capsids. Transduction profiles of three retinas injected with CMV-GFP vectors packaged in AAV6, ShH10, and ShH10Y capsids at (d) 108, (e) 109, and (f) 1010 gc showed that ShH10Y is the most powerful capsid to transduce mouse Müller glial cells even at low doses. Data are presented as mean ± SEM and n = 3/AAV/dose. **P < 0.01, ***P < 0.001. Bar = 50 µm (a–c). AAV, adeno-associated virus; CMV, cytomegalovirus; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PRC, photoreceptor cell; RPE, retinal pigment epithelium.
Mentions: We studied the transduction and expression profiles of AAV6, ShH10, and ShH10Y, (ShH10Y carries an additional tyrosine to phenylalanine mutation to enhance its transduction efficiency).21 We intravitreally injected Crb1−/− retinas with 108, 109, 1010 genome copies (gc) of CMV-GFP transgene packaged in the different AAV serotypes. We analyzed the GFP expression by scanning laser ophthalmoscopy (SLO) and immunohistochemistry using cell type–specific immunomarkers in retinal sections. At 108 gc, the ShH10Y variant showed enhanced ability to transduce Müller glial cells relative to the unmodified ShH10 and AAV6 capsids (49 ± 6, 24 ± 3, and 21 ± 3%, respectively; Figure 1d), the total number of GFP-positive cells per millimeter was not different (28 ± 4, 29 ± 6, and 26 ± 3%, respectively). At one log unit higher titer (109 gc), ShH10Y also showed the highest percentage of transduced mouse Müller glial cells, whereas ShH10 and AAV6 transduced less efficiently (66 ± 4, 23 ± 2, and 17 ± 6%, respectively; Figure 1e), the total number of infected cells per millimeter was not different (49 ± 2, 47 ± 10, and 51 ± 2, respectively). At 1010 gc, the cell types infected by AAV6 were primarily around the blood vessels as shown previously,14 whereas both ShH10 and ShH10Y showed a broader transduction pattern (Figure 1a–c). 62 ± 2% of the cells transduced by ShH10Y were Müller glial cells, whereas significantly less Müller glial cells were transduced with ShH10 (39 ± 1%) or AAV6 (18 ± 1%) (Figure 1a–c,f). The number of GFP-positive cells per millimeter was not significantly different (152 ± 25, 123 ± 44, and 147 ± 17, respectively). In summary, intravitreally injected ShH10Y vectors showed higher transduction efficiency for mouse Müller glial cells than AAV6 or ShH10 vectors.

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