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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

ShH10 and ShH10Y variants transduced human Müller glial cells in vitro. Transverse section of cultured retinas from donor eyes with 1010 genome copies of (a,b) AAV2/ShH10-CMV-GFP, (c) AAV2/ShH10Y-RLBP1-GFP, and (d) AAV2/ShH10Y-CMV-GFP revealed the presence of GFP-positive Müller glial cells with the three different vectors (a,c,d) and GFP-positive photoreceptors only with CMV promoter vectors. Bar: 50 µm (a–c). AAV, adeno-associated virus; CMV, cytomegalovirus; GFP, green fluorescent protein; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RLBP1, retinaldehyde binding protein 1.
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fig6: ShH10 and ShH10Y variants transduced human Müller glial cells in vitro. Transverse section of cultured retinas from donor eyes with 1010 genome copies of (a,b) AAV2/ShH10-CMV-GFP, (c) AAV2/ShH10Y-RLBP1-GFP, and (d) AAV2/ShH10Y-CMV-GFP revealed the presence of GFP-positive Müller glial cells with the three different vectors (a,c,d) and GFP-positive photoreceptors only with CMV promoter vectors. Bar: 50 µm (a–c). AAV, adeno-associated virus; CMV, cytomegalovirus; GFP, green fluorescent protein; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RLBP1, retinaldehyde binding protein 1.

Mentions: We tested the potency of ShH10 and ShH10Y capsids and RLBP1 promoter to transduce and express GFP in human Müller glial cells. For this purpose, application of 1010 gc of ShH10-CMV-GFP, ShH10Y-CMV-GFP, and ShH10Y-RLBP1-GFP was performed on cultures of human retinal explants. After 7 days in culture, transverse retinal sections revealed GFP expression in human Müller glial cells independently of the capsids or promoter used (Figure 6a,c,d). Retinas injected with CMV-GFP vectors showed many transduced photoreceptors at the site of injection, whereas no transduced photoreceptors were detected with RLBP1-GFP (Figure 6b).


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)

ShH10 and ShH10Y variants transduced human Müller glial cells in vitro. Transverse section of cultured retinas from donor eyes with 1010 genome copies of (a,b) AAV2/ShH10-CMV-GFP, (c) AAV2/ShH10Y-RLBP1-GFP, and (d) AAV2/ShH10Y-CMV-GFP revealed the presence of GFP-positive Müller glial cells with the three different vectors (a,c,d) and GFP-positive photoreceptors only with CMV promoter vectors. Bar: 50 µm (a–c). AAV, adeno-associated virus; CMV, cytomegalovirus; GFP, green fluorescent protein; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RLBP1, retinaldehyde binding protein 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362388&req=5

fig6: ShH10 and ShH10Y variants transduced human Müller glial cells in vitro. Transverse section of cultured retinas from donor eyes with 1010 genome copies of (a,b) AAV2/ShH10-CMV-GFP, (c) AAV2/ShH10Y-RLBP1-GFP, and (d) AAV2/ShH10Y-CMV-GFP revealed the presence of GFP-positive Müller glial cells with the three different vectors (a,c,d) and GFP-positive photoreceptors only with CMV promoter vectors. Bar: 50 µm (a–c). AAV, adeno-associated virus; CMV, cytomegalovirus; GFP, green fluorescent protein; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RLBP1, retinaldehyde binding protein 1.
Mentions: We tested the potency of ShH10 and ShH10Y capsids and RLBP1 promoter to transduce and express GFP in human Müller glial cells. For this purpose, application of 1010 gc of ShH10-CMV-GFP, ShH10Y-CMV-GFP, and ShH10Y-RLBP1-GFP was performed on cultures of human retinal explants. After 7 days in culture, transverse retinal sections revealed GFP expression in human Müller glial cells independently of the capsids or promoter used (Figure 6a,c,d). Retinas injected with CMV-GFP vectors showed many transduced photoreceptors at the site of injection, whereas no transduced photoreceptors were detected with RLBP1-GFP (Figure 6b).

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