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Long-term and age-dependent restoration of visual function in a mouse model of CNGB3-associated achromatopsia following gene therapy.

Carvalho LS, Xu J, Pearson RA, Smith AJ, Bainbridge JW, Morris LM, Fliesler SJ, Ding XQ, Ali RR - Hum. Mol. Genet. (2011)

Bottom Line: Following subretinal delivery of the vector, CNGB3 was detected in both M- and S-cones and resulted in increased levels of CNGA3, increased cone density and survival, improved cone outer segment structure and normal subcellular compartmentalization of cone opsins.Therapy also resulted in long-term improvement of retinal function, with restoration of cone ERG amplitudes of up to 90% of wild-type and a significant improvement in visual acuity.Remarkably, successful restoration of cone function was observed even when treatment was initiated at 6 months of age; however, restoration of normal visual acuity was only possible in younger animals (e.g. 2-4 weeks old).

View Article: PubMed Central - PubMed

Affiliation: The Department of Genetics, UCL Institute of Ophthalmology, London, UK.

ABSTRACT
Mutations in the CNGB3 gene account for >50% of all known cases of achromatopsia. Although of early onset, its stationary character and the potential for rapid assessment of restoration of retinal function following therapy renders achromatopsia a very attractive candidate for gene therapy. Here we tested the efficacy of an rAAV2/8 vector containing a human cone arrestin promoter and a human CNGB3 cDNA in CNGB3 deficient mice. Following subretinal delivery of the vector, CNGB3 was detected in both M- and S-cones and resulted in increased levels of CNGA3, increased cone density and survival, improved cone outer segment structure and normal subcellular compartmentalization of cone opsins. Therapy also resulted in long-term improvement of retinal function, with restoration of cone ERG amplitudes of up to 90% of wild-type and a significant improvement in visual acuity. Remarkably, successful restoration of cone function was observed even when treatment was initiated at 6 months of age; however, restoration of normal visual acuity was only possible in younger animals (e.g. 2-4 weeks old). This study represents achievement of the most substantial restoration of visual function reported to date in an animal model of achromatopsia using a human gene construct, which has the potential to be utilized in clinical trials.

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Related in: MedlinePlus

Subretinal injection of AAV2/8 vector containing human cone arrestin promoter results in cone-specific transgenic expression in Cngb3−/− mice. Injections were performed at P15 and eyes were collected at 30 days PI to analyze expression of CNGB3. Immunohistochemical detection of CNGB3 expression in the treated (a, b, c) and untreated (d, e, f) Cngb3−/− mice. Retinal cross-sections were immunolabeled for CNGB3 (green), S-opsin (red), M-opsin (red) with a nuclear counterstain (DAPI, blue). c′ and f′ are higher magnification images of treated and untreated overlay. Shown are representative images of the analyses from 4 to 6 mice/group. OS, outer segment; ONL, outer nuclear layer; and INL, inner nuclear layer. Scale bars = 50 μm.
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DDR218F1: Subretinal injection of AAV2/8 vector containing human cone arrestin promoter results in cone-specific transgenic expression in Cngb3−/− mice. Injections were performed at P15 and eyes were collected at 30 days PI to analyze expression of CNGB3. Immunohistochemical detection of CNGB3 expression in the treated (a, b, c) and untreated (d, e, f) Cngb3−/− mice. Retinal cross-sections were immunolabeled for CNGB3 (green), S-opsin (red), M-opsin (red) with a nuclear counterstain (DAPI, blue). c′ and f′ are higher magnification images of treated and untreated overlay. Shown are representative images of the analyses from 4 to 6 mice/group. OS, outer segment; ONL, outer nuclear layer; and INL, inner nuclear layer. Scale bars = 50 μm.

Mentions: We constructed a recombinant AAV2/8 viral vector containing a 0.4 kb fragment of the human cone arrestin promoter (hCAR) and the human CNGB3 cDNA (rAAV2/8_hCAR_hCNGB3). The vector was injected subretinally into Cngb3−/− mice at post-natal day 15 (P15). Eyes were isolated at 30 days post-injection (PI) and expression of CNGB3 was evaluated by immunohistochemistry. As shown in Figure 1, CNGB3 immunoreactivity was detected in the outer segments in retinal sections prepared from eyes that had received vector, but not in the uninjected controls. Cone-specific CNGB3 transgene expression was demonstrated in both M- and S-cones by co-labeling with the antibodies against M-opsin and S-opsin, respectively (Fig. 1).Figure 1.


Long-term and age-dependent restoration of visual function in a mouse model of CNGB3-associated achromatopsia following gene therapy.

Carvalho LS, Xu J, Pearson RA, Smith AJ, Bainbridge JW, Morris LM, Fliesler SJ, Ding XQ, Ali RR - Hum. Mol. Genet. (2011)

Subretinal injection of AAV2/8 vector containing human cone arrestin promoter results in cone-specific transgenic expression in Cngb3−/− mice. Injections were performed at P15 and eyes were collected at 30 days PI to analyze expression of CNGB3. Immunohistochemical detection of CNGB3 expression in the treated (a, b, c) and untreated (d, e, f) Cngb3−/− mice. Retinal cross-sections were immunolabeled for CNGB3 (green), S-opsin (red), M-opsin (red) with a nuclear counterstain (DAPI, blue). c′ and f′ are higher magnification images of treated and untreated overlay. Shown are representative images of the analyses from 4 to 6 mice/group. OS, outer segment; ONL, outer nuclear layer; and INL, inner nuclear layer. Scale bars = 50 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3140821&req=5

DDR218F1: Subretinal injection of AAV2/8 vector containing human cone arrestin promoter results in cone-specific transgenic expression in Cngb3−/− mice. Injections were performed at P15 and eyes were collected at 30 days PI to analyze expression of CNGB3. Immunohistochemical detection of CNGB3 expression in the treated (a, b, c) and untreated (d, e, f) Cngb3−/− mice. Retinal cross-sections were immunolabeled for CNGB3 (green), S-opsin (red), M-opsin (red) with a nuclear counterstain (DAPI, blue). c′ and f′ are higher magnification images of treated and untreated overlay. Shown are representative images of the analyses from 4 to 6 mice/group. OS, outer segment; ONL, outer nuclear layer; and INL, inner nuclear layer. Scale bars = 50 μm.
Mentions: We constructed a recombinant AAV2/8 viral vector containing a 0.4 kb fragment of the human cone arrestin promoter (hCAR) and the human CNGB3 cDNA (rAAV2/8_hCAR_hCNGB3). The vector was injected subretinally into Cngb3−/− mice at post-natal day 15 (P15). Eyes were isolated at 30 days post-injection (PI) and expression of CNGB3 was evaluated by immunohistochemistry. As shown in Figure 1, CNGB3 immunoreactivity was detected in the outer segments in retinal sections prepared from eyes that had received vector, but not in the uninjected controls. Cone-specific CNGB3 transgene expression was demonstrated in both M- and S-cones by co-labeling with the antibodies against M-opsin and S-opsin, respectively (Fig. 1).Figure 1.

Bottom Line: Following subretinal delivery of the vector, CNGB3 was detected in both M- and S-cones and resulted in increased levels of CNGA3, increased cone density and survival, improved cone outer segment structure and normal subcellular compartmentalization of cone opsins.Therapy also resulted in long-term improvement of retinal function, with restoration of cone ERG amplitudes of up to 90% of wild-type and a significant improvement in visual acuity.Remarkably, successful restoration of cone function was observed even when treatment was initiated at 6 months of age; however, restoration of normal visual acuity was only possible in younger animals (e.g. 2-4 weeks old).

View Article: PubMed Central - PubMed

Affiliation: The Department of Genetics, UCL Institute of Ophthalmology, London, UK.

ABSTRACT
Mutations in the CNGB3 gene account for >50% of all known cases of achromatopsia. Although of early onset, its stationary character and the potential for rapid assessment of restoration of retinal function following therapy renders achromatopsia a very attractive candidate for gene therapy. Here we tested the efficacy of an rAAV2/8 vector containing a human cone arrestin promoter and a human CNGB3 cDNA in CNGB3 deficient mice. Following subretinal delivery of the vector, CNGB3 was detected in both M- and S-cones and resulted in increased levels of CNGA3, increased cone density and survival, improved cone outer segment structure and normal subcellular compartmentalization of cone opsins. Therapy also resulted in long-term improvement of retinal function, with restoration of cone ERG amplitudes of up to 90% of wild-type and a significant improvement in visual acuity. Remarkably, successful restoration of cone function was observed even when treatment was initiated at 6 months of age; however, restoration of normal visual acuity was only possible in younger animals (e.g. 2-4 weeks old). This study represents achievement of the most substantial restoration of visual function reported to date in an animal model of achromatopsia using a human gene construct, which has the potential to be utilized in clinical trials.

Show MeSH
Related in: MedlinePlus