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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

Vector-mediated improvement of dark adaptation in Rlbp1-/- mice. (a–d) ERGs were measured in mice fully dark-adapted overnight (>15 hours) to obtain responses to a single intensity white light flash. Following another overnight dark adaptation, mice were exposed to a bleaching light, dark adapted for 4 hours and ERGs were remeasured. (a,b) ERG traces are shown from (a) 70-week-old naive Rlbp1+/+ and (b) 68-week-old naive Rlbp1-/- mice recorded in conjunction with the week 50 postinjection measurement shown in Figure 6. The maximum responses after full dark adaptation are shown as bold lines while responses 4 hours following a photobleach are shown as thin lines. Arrows point to the a-wave of the ERG responses. (c) ERGs from naive and treated Rlbp1-/- mice 4 hours following a photobleach. The recordings were performed 50 weeks postsubretinal injection. Note that the time scale is expanded compared to (a) and (b). The open triangle (treated) and open circle (naive) symbols indicate the time after a flash (5 milliseconds) at which we measured amplitudes. (d) The graph contains 50-week postinjection a-wave recovery data (dark adaptation) from all tested mice achieved by dividing the a-wave amplitude 4 hours postphotobleach in each eye by its respective maximum dark-adapted value (same data set as presented in Figure 6, 50-week time point for AAV8 vector treated and naive eyes). Open symbols in (d) correspond to data from the traces in (c). Each symbol represents 1 eye of each mouse tested. AAV8, subretinally injected with scAAV8-pRLBP1(short)-hRLBP1. Calculations of P values compare treated eyes to naive Rlbp1-/- eyes. ****P ≤ 0.0001.
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fig4: Vector-mediated improvement of dark adaptation in Rlbp1-/- mice. (a–d) ERGs were measured in mice fully dark-adapted overnight (>15 hours) to obtain responses to a single intensity white light flash. Following another overnight dark adaptation, mice were exposed to a bleaching light, dark adapted for 4 hours and ERGs were remeasured. (a,b) ERG traces are shown from (a) 70-week-old naive Rlbp1+/+ and (b) 68-week-old naive Rlbp1-/- mice recorded in conjunction with the week 50 postinjection measurement shown in Figure 6. The maximum responses after full dark adaptation are shown as bold lines while responses 4 hours following a photobleach are shown as thin lines. Arrows point to the a-wave of the ERG responses. (c) ERGs from naive and treated Rlbp1-/- mice 4 hours following a photobleach. The recordings were performed 50 weeks postsubretinal injection. Note that the time scale is expanded compared to (a) and (b). The open triangle (treated) and open circle (naive) symbols indicate the time after a flash (5 milliseconds) at which we measured amplitudes. (d) The graph contains 50-week postinjection a-wave recovery data (dark adaptation) from all tested mice achieved by dividing the a-wave amplitude 4 hours postphotobleach in each eye by its respective maximum dark-adapted value (same data set as presented in Figure 6, 50-week time point for AAV8 vector treated and naive eyes). Open symbols in (d) correspond to data from the traces in (c). Each symbol represents 1 eye of each mouse tested. AAV8, subretinally injected with scAAV8-pRLBP1(short)-hRLBP1. Calculations of P values compare treated eyes to naive Rlbp1-/- eyes. ****P ≤ 0.0001.

Mentions: Similar to young patients with RLBP1-associated retinitis pigmentosa prior to substantial retinal degeneration, after long periods of dark adaption (e.g., ≥24 hours), the ERGs of Rlbp1-/- mice had normal amplitudes in response to single flashes of light.10 However, after bleaches of more than 10% of rhodopsin, Rlbp1-/- mice required many hours of dark adaptation to regain full light sensitivity, in contrast to the substantial recovery observed in only about 3 hours in Rlbp1+/+ mice.10Figure 4a,b illustrates the difference in ERG amplitudes between Rlbp1-/- and Rlbp1+/+ mice after 4 hours of dark adaptation as compared with the fully dark-adapted responses of each mouse. Rlbp1+/+ mice exhibit nearly full recovery of scotopic visual function (as indicated by a-wave amplitudes) in contrast to Rlbp1-/- mice which have minimal recovery. This slow dark adaptation is in qualitative agreement with results reported previously in Rlbp1-/- mice with a different mutation.10


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)

Vector-mediated improvement of dark adaptation in Rlbp1-/- mice. (a–d) ERGs were measured in mice fully dark-adapted overnight (>15 hours) to obtain responses to a single intensity white light flash. Following another overnight dark adaptation, mice were exposed to a bleaching light, dark adapted for 4 hours and ERGs were remeasured. (a,b) ERG traces are shown from (a) 70-week-old naive Rlbp1+/+ and (b) 68-week-old naive Rlbp1-/- mice recorded in conjunction with the week 50 postinjection measurement shown in Figure 6. The maximum responses after full dark adaptation are shown as bold lines while responses 4 hours following a photobleach are shown as thin lines. Arrows point to the a-wave of the ERG responses. (c) ERGs from naive and treated Rlbp1-/- mice 4 hours following a photobleach. The recordings were performed 50 weeks postsubretinal injection. Note that the time scale is expanded compared to (a) and (b). The open triangle (treated) and open circle (naive) symbols indicate the time after a flash (5 milliseconds) at which we measured amplitudes. (d) The graph contains 50-week postinjection a-wave recovery data (dark adaptation) from all tested mice achieved by dividing the a-wave amplitude 4 hours postphotobleach in each eye by its respective maximum dark-adapted value (same data set as presented in Figure 6, 50-week time point for AAV8 vector treated and naive eyes). Open symbols in (d) correspond to data from the traces in (c). Each symbol represents 1 eye of each mouse tested. AAV8, subretinally injected with scAAV8-pRLBP1(short)-hRLBP1. Calculations of P values compare treated eyes to naive Rlbp1-/- eyes. ****P ≤ 0.0001.
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Related In: Results  -  Collection

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fig4: Vector-mediated improvement of dark adaptation in Rlbp1-/- mice. (a–d) ERGs were measured in mice fully dark-adapted overnight (>15 hours) to obtain responses to a single intensity white light flash. Following another overnight dark adaptation, mice were exposed to a bleaching light, dark adapted for 4 hours and ERGs were remeasured. (a,b) ERG traces are shown from (a) 70-week-old naive Rlbp1+/+ and (b) 68-week-old naive Rlbp1-/- mice recorded in conjunction with the week 50 postinjection measurement shown in Figure 6. The maximum responses after full dark adaptation are shown as bold lines while responses 4 hours following a photobleach are shown as thin lines. Arrows point to the a-wave of the ERG responses. (c) ERGs from naive and treated Rlbp1-/- mice 4 hours following a photobleach. The recordings were performed 50 weeks postsubretinal injection. Note that the time scale is expanded compared to (a) and (b). The open triangle (treated) and open circle (naive) symbols indicate the time after a flash (5 milliseconds) at which we measured amplitudes. (d) The graph contains 50-week postinjection a-wave recovery data (dark adaptation) from all tested mice achieved by dividing the a-wave amplitude 4 hours postphotobleach in each eye by its respective maximum dark-adapted value (same data set as presented in Figure 6, 50-week time point for AAV8 vector treated and naive eyes). Open symbols in (d) correspond to data from the traces in (c). Each symbol represents 1 eye of each mouse tested. AAV8, subretinally injected with scAAV8-pRLBP1(short)-hRLBP1. Calculations of P values compare treated eyes to naive Rlbp1-/- eyes. ****P ≤ 0.0001.
Mentions: Similar to young patients with RLBP1-associated retinitis pigmentosa prior to substantial retinal degeneration, after long periods of dark adaption (e.g., ≥24 hours), the ERGs of Rlbp1-/- mice had normal amplitudes in response to single flashes of light.10 However, after bleaches of more than 10% of rhodopsin, Rlbp1-/- mice required many hours of dark adaptation to regain full light sensitivity, in contrast to the substantial recovery observed in only about 3 hours in Rlbp1+/+ mice.10Figure 4a,b illustrates the difference in ERG amplitudes between Rlbp1-/- and Rlbp1+/+ mice after 4 hours of dark adaptation as compared with the fully dark-adapted responses of each mouse. Rlbp1+/+ mice exhibit nearly full recovery of scotopic visual function (as indicated by a-wave amplitudes) in contrast to Rlbp1-/- mice which have minimal recovery. This slow dark adaptation is in qualitative agreement with results reported previously in Rlbp1-/- mice with a different mutation.10

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