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AAV-mediated RLBP1 gene therapy improves the rate of dark adaptation in Rlbp1 knockout mice.

Choi VW, Bigelow CE, McGee TL, Gujar AN, Li H, Hanks SM, Vrouvlianis J, Maker M, Leehy B, Zhang Y, Aranda J, Bounoutas G, Demirs JT, Yang J, Ornberg R, Wang Y, Martin W, Stout KR, Argentieri G, Grosenstein P, Diaz D, Turner O, Jaffee BD, Police SR, Dryja TP - Mol Ther Methods Clin Dev (2015)

Bottom Line: We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein).The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome.The effect was still present after 1 year.

View Article: PubMed Central - PubMed

Affiliation: Ophthalmology Disease Area, Novartis Institutes for BioMedical Research , Cambridge, Massachusetts, USA.

ABSTRACT
Recessive mutations in RLBP1 cause a form of retinitis pigmentosa in which the retina, before its degeneration leads to blindness, abnormally slowly recovers sensitivity after exposure to light. To develop a potential gene therapy for this condition, we tested multiple recombinant adeno-associated vectors (rAAVs) composed of different promoters, capsid serotypes, and genome conformations. We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein). A promoter derived from the RLBP1 gene mediated expression in the retinal pigment epithelium and Müller cells (the intended target cell types) at qualitatively higher levels than in other retinal cell types in wild-type mice and monkeys. With this promoter upstream of the coding sequence of the human RLBP1 gene, we compared the potencies of vectors with an AAV2 versus an AAV8 capsid in transducing mouse retinas, and we compared vectors with a self-complementary versus a single-stranded genome. The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome. Subretinal injection of scAAV8-pRLBP1-hRLBP1 in Rlbp1 izygous mice improved the rate of dark adaptation based on scotopic (rod-plus-cone) and photopic (cone) electroretinograms (ERGs). The effect was still present after 1 year.

No MeSH data available.


Related in: MedlinePlus

Dose–response relationship of a-wave recoveries in Rlbp1-/- mice treated with scAAV8-pRLBP1(short)-hRLBP1. Data points represent a-wave recoveries of individual mouse eyes (1 eye per mouse) assessed 12 or 13 weeks postsubretinal delivery of 3 × 106, 3 × 107, 3 × 108, or 1 × 109 vg/eye of scAAV8-pRLBP1(short)-hRLBP1 or 1 × 109 vg/eye of a  vector. The 95% confidence interval (CI) of the mean from several cohorts of Rlbp1+/+ mice is represented by the shaded region (n = 23 eyes). Calculations of p-values compare treated eyes to naive Rlbp1-/- eyes. ***P ≤ 0.001; ****P ≤ 0.0001.
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fig5: Dose–response relationship of a-wave recoveries in Rlbp1-/- mice treated with scAAV8-pRLBP1(short)-hRLBP1. Data points represent a-wave recoveries of individual mouse eyes (1 eye per mouse) assessed 12 or 13 weeks postsubretinal delivery of 3 × 106, 3 × 107, 3 × 108, or 1 × 109 vg/eye of scAAV8-pRLBP1(short)-hRLBP1 or 1 × 109 vg/eye of a vector. The 95% confidence interval (CI) of the mean from several cohorts of Rlbp1+/+ mice is represented by the shaded region (n = 23 eyes). Calculations of p-values compare treated eyes to naive Rlbp1-/- eyes. ***P ≤ 0.001; ****P ≤ 0.0001.

Mentions: We explored the dose-response relationship of the vector by administering subretinal doses of 3 × 106, 3 × 107, 3 × 108, and 1 × 109 vg/eye. For comparison, negative controls included naive mice as well as mice that received injections of a rAAV8 vector carrying no transgene at a dose of 1 × 109 vg/eye. After four hours of dark adaptation, mouse eyes that had received scAAV8-pRLBP1(short)-hRLBP1 at doses of 3 × 108 and 1 × 109 vg/eye had ERG amplitudes 69 and 85%, respectively, of the maximum amplitudes achieved after overnight dark adaptation. The a-wave recoveries of these eyes were greater than those eyes receiving lower doses, negative control vector, or were untreated (Figure 5).


AAV-mediated RLBP1 gene therapy improves the rate of dark adaptation in Rlbp1 knockout mice.

Choi VW, Bigelow CE, McGee TL, Gujar AN, Li H, Hanks SM, Vrouvlianis J, Maker M, Leehy B, Zhang Y, Aranda J, Bounoutas G, Demirs JT, Yang J, Ornberg R, Wang Y, Martin W, Stout KR, Argentieri G, Grosenstein P, Diaz D, Turner O, Jaffee BD, Police SR, Dryja TP - Mol Ther Methods Clin Dev (2015)

Dose–response relationship of a-wave recoveries in Rlbp1-/- mice treated with scAAV8-pRLBP1(short)-hRLBP1. Data points represent a-wave recoveries of individual mouse eyes (1 eye per mouse) assessed 12 or 13 weeks postsubretinal delivery of 3 × 106, 3 × 107, 3 × 108, or 1 × 109 vg/eye of scAAV8-pRLBP1(short)-hRLBP1 or 1 × 109 vg/eye of a  vector. The 95% confidence interval (CI) of the mean from several cohorts of Rlbp1+/+ mice is represented by the shaded region (n = 23 eyes). Calculations of p-values compare treated eyes to naive Rlbp1-/- eyes. ***P ≤ 0.001; ****P ≤ 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495722&req=5

fig5: Dose–response relationship of a-wave recoveries in Rlbp1-/- mice treated with scAAV8-pRLBP1(short)-hRLBP1. Data points represent a-wave recoveries of individual mouse eyes (1 eye per mouse) assessed 12 or 13 weeks postsubretinal delivery of 3 × 106, 3 × 107, 3 × 108, or 1 × 109 vg/eye of scAAV8-pRLBP1(short)-hRLBP1 or 1 × 109 vg/eye of a vector. The 95% confidence interval (CI) of the mean from several cohorts of Rlbp1+/+ mice is represented by the shaded region (n = 23 eyes). Calculations of p-values compare treated eyes to naive Rlbp1-/- eyes. ***P ≤ 0.001; ****P ≤ 0.0001.
Mentions: We explored the dose-response relationship of the vector by administering subretinal doses of 3 × 106, 3 × 107, 3 × 108, and 1 × 109 vg/eye. For comparison, negative controls included naive mice as well as mice that received injections of a rAAV8 vector carrying no transgene at a dose of 1 × 109 vg/eye. After four hours of dark adaptation, mouse eyes that had received scAAV8-pRLBP1(short)-hRLBP1 at doses of 3 × 108 and 1 × 109 vg/eye had ERG amplitudes 69 and 85%, respectively, of the maximum amplitudes achieved after overnight dark adaptation. The a-wave recoveries of these eyes were greater than those eyes receiving lower doses, negative control vector, or were untreated (Figure 5).

Bottom Line: We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein).The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome.The effect was still present after 1 year.

View Article: PubMed Central - PubMed

Affiliation: Ophthalmology Disease Area, Novartis Institutes for BioMedical Research , Cambridge, Massachusetts, USA.

ABSTRACT
Recessive mutations in RLBP1 cause a form of retinitis pigmentosa in which the retina, before its degeneration leads to blindness, abnormally slowly recovers sensitivity after exposure to light. To develop a potential gene therapy for this condition, we tested multiple recombinant adeno-associated vectors (rAAVs) composed of different promoters, capsid serotypes, and genome conformations. We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein). A promoter derived from the RLBP1 gene mediated expression in the retinal pigment epithelium and Müller cells (the intended target cell types) at qualitatively higher levels than in other retinal cell types in wild-type mice and monkeys. With this promoter upstream of the coding sequence of the human RLBP1 gene, we compared the potencies of vectors with an AAV2 versus an AAV8 capsid in transducing mouse retinas, and we compared vectors with a self-complementary versus a single-stranded genome. The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome. Subretinal injection of scAAV8-pRLBP1-hRLBP1 in Rlbp1 izygous mice improved the rate of dark adaptation based on scotopic (rod-plus-cone) and photopic (cone) electroretinograms (ERGs). The effect was still present after 1 year.

No MeSH data available.


Related in: MedlinePlus