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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, ShH10Y, and AAV9 tropism following subretinal injection in adult murine retina. Representative transverse retinal section of Crb1−/− mice 3 weeks after subretinal injection of 109 genome copies (gc) of CMV-GFP with (a) ShH10, (b) ShH10Y, and (c) AAV9 capsids showed more GFP-positive cells with AAV9 capsids. (d) Transduction profiles of three retinas injected with CMV-GFP vectors packaged in AAV6, ShH10, ShH10Y, and AAV9 revealed that ShH10Y and AAV9 were the most powerful capsids to transduce mouse Müller glial cells via the subretinal route in contrast to AAV6 and ShH10. Data are presented as mean ± SEM and n = 3/AAV/dose. *P < 0.05. Bar: 50 µm (a–c). AAV, adeno-associated virus; CMV, cytomegalovirus; GCL, ganglion cell layer; GFP, green fluorescent protein; INL, inner nuclear layer; ONL, outer nuclear layer; PRC, photoreceptor cell; RPE, retinal pigment epithelium.
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fig2: ShH10, ShH10Y, and AAV9 tropism following subretinal injection in adult murine retina. Representative transverse retinal section of Crb1−/− mice 3 weeks after subretinal injection of 109 genome copies (gc) of CMV-GFP with (a) ShH10, (b) ShH10Y, and (c) AAV9 capsids showed more GFP-positive cells with AAV9 capsids. (d) Transduction profiles of three retinas injected with CMV-GFP vectors packaged in AAV6, ShH10, ShH10Y, and AAV9 revealed that ShH10Y and AAV9 were the most powerful capsids to transduce mouse Müller glial cells via the subretinal route in contrast to AAV6 and ShH10. Data are presented as mean ± SEM and n = 3/AAV/dose. *P < 0.05. Bar: 50 µm (a–c). AAV, adeno-associated virus; CMV, cytomegalovirus; GCL, ganglion cell layer; GFP, green fluorescent protein; INL, inner nuclear layer; ONL, outer nuclear layer; PRC, photoreceptor cell; RPE, retinal pigment epithelium.

Mentions: The AAV9 capsid has been shown to efficiently transduce many retinal cells including photoreceptors and Müller glial cells via the subretinal route.22,23 Intravitreal injection of AAV9-CMV-GFP resulted in transduction of few ganglion cells (data not shown). We compared the subretinal transduction efficiency of AAV9 with AAV6,14 ShH10, and ShH10Y (Figure 2). The percentage of GFP-positive Müller glial cells produced by AAV9 and ShH10Y injection was higher and similar (30 ± 2 and 26 ± 6%, respectively) relative to AAV6 and ShH10 vectors (11 ± 1 and 7 ± 3%, respectively). However, AAV9 transduced more mouse retinal cells than ShH10Y (483 ± 171 versus 100 ± 33 GFP-positive cells per millimeter). In summary, AAV9 and ShH10Y are potent capsids to transduce mouse Müller glial cells via the subretinal route.


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, ShH10Y, and AAV9 tropism following subretinal injection in adult murine retina. Representative transverse retinal section of Crb1−/− mice 3 weeks after subretinal injection of 109 genome copies (gc) of CMV-GFP with (a) ShH10, (b) ShH10Y, and (c) AAV9 capsids showed more GFP-positive cells with AAV9 capsids. (d) Transduction profiles of three retinas injected with CMV-GFP vectors packaged in AAV6, ShH10, ShH10Y, and AAV9 revealed that ShH10Y and AAV9 were the most powerful capsids to transduce mouse Müller glial cells via the subretinal route in contrast to AAV6 and ShH10. Data are presented as mean ± SEM and n = 3/AAV/dose. *P < 0.05. Bar: 50 µm (a–c). AAV, adeno-associated virus; CMV, cytomegalovirus; GCL, ganglion cell layer; GFP, green fluorescent protein; INL, inner nuclear layer; ONL, outer nuclear layer; PRC, photoreceptor cell; RPE, retinal pigment epithelium.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: ShH10, ShH10Y, and AAV9 tropism following subretinal injection in adult murine retina. Representative transverse retinal section of Crb1−/− mice 3 weeks after subretinal injection of 109 genome copies (gc) of CMV-GFP with (a) ShH10, (b) ShH10Y, and (c) AAV9 capsids showed more GFP-positive cells with AAV9 capsids. (d) Transduction profiles of three retinas injected with CMV-GFP vectors packaged in AAV6, ShH10, ShH10Y, and AAV9 revealed that ShH10Y and AAV9 were the most powerful capsids to transduce mouse Müller glial cells via the subretinal route in contrast to AAV6 and ShH10. Data are presented as mean ± SEM and n = 3/AAV/dose. *P < 0.05. Bar: 50 µm (a–c). AAV, adeno-associated virus; CMV, cytomegalovirus; GCL, ganglion cell layer; GFP, green fluorescent protein; INL, inner nuclear layer; ONL, outer nuclear layer; PRC, photoreceptor cell; RPE, retinal pigment epithelium.
Mentions: The AAV9 capsid has been shown to efficiently transduce many retinal cells including photoreceptors and Müller glial cells via the subretinal route.22,23 Intravitreal injection of AAV9-CMV-GFP resulted in transduction of few ganglion cells (data not shown). We compared the subretinal transduction efficiency of AAV9 with AAV6,14 ShH10, and ShH10Y (Figure 2). The percentage of GFP-positive Müller glial cells produced by AAV9 and ShH10Y injection was higher and similar (30 ± 2 and 26 ± 6%, respectively) relative to AAV6 and ShH10 vectors (11 ± 1 and 7 ± 3%, respectively). However, AAV9 transduced more mouse retinal cells than ShH10Y (483 ± 171 versus 100 ± 33 GFP-positive cells per millimeter). In summary, AAV9 and ShH10Y are potent capsids to transduce mouse Müller glial cells via the subretinal route.

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