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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

scAAV8-pRLBP1(short)-hRLBP1 improves cone dark adaptation in Rlbp1-/- mice. (a–c) Cone-driven ERG-responses were extracted by the measurement of b-wave amplitudes from a paired-flash protocol at various time points following a 1 minute photobleach. Representative ERG traces from the paired-flash protocol at 0.0, 2.5, and 5.0 minutes post-bleach in Rlbp1+/+ mice, Rlbp1-/- mice, and Rlbp1-/- mice treated with a subretinal injection scAAV8-pRLBP1(short)-hRLBP1 10 weeks prior to recordings are shown (a–c respectively). (d) The graph displays average b-wave recoveries normalized to dark-adapted pre-bleach levels in Rlbp1+/+ (n = 4), Rlbp1-/- (n = 3), and vector treated Rlbp1-/- (n = 9) mice. The P values displayed in the plot compare the b-wave recovery at the 0 minute time point to later time points within the AAV8 vector-treated group (***P ≤ 0.001; ****P ≤ 0.0001). Analysis was also performed to compare recoveries between groups at each time point (not displayed in the plot). This comparison indicates significant recovery in naive Rlbp1+/+ mice and Rlbp1-/- mice receiving AAV8 vector compared to naive Rlbp1-/- mice at 2.5, 5, and 20 minutes postbleach (naive Rlbp1+/+: P ≤ 0.05, P ≤ 0.01, P ≤ 0.05; Rlbp1-/- + AAV8 vector. P ≤ 0.01, P ≤ 0.01, P ≤ 0.01 for the respective 2.5, 5, and 20 minute time points). No corresponding difference was detected at 0, 10, or 15 minutes (P > 0.05). No significant difference was detected between Rlbp1+/+ and Rlbp1-/- + AAV8 vector groups at any time point.
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fig7: scAAV8-pRLBP1(short)-hRLBP1 improves cone dark adaptation in Rlbp1-/- mice. (a–c) Cone-driven ERG-responses were extracted by the measurement of b-wave amplitudes from a paired-flash protocol at various time points following a 1 minute photobleach. Representative ERG traces from the paired-flash protocol at 0.0, 2.5, and 5.0 minutes post-bleach in Rlbp1+/+ mice, Rlbp1-/- mice, and Rlbp1-/- mice treated with a subretinal injection scAAV8-pRLBP1(short)-hRLBP1 10 weeks prior to recordings are shown (a–c respectively). (d) The graph displays average b-wave recoveries normalized to dark-adapted pre-bleach levels in Rlbp1+/+ (n = 4), Rlbp1-/- (n = 3), and vector treated Rlbp1-/- (n = 9) mice. The P values displayed in the plot compare the b-wave recovery at the 0 minute time point to later time points within the AAV8 vector-treated group (***P ≤ 0.001; ****P ≤ 0.0001). Analysis was also performed to compare recoveries between groups at each time point (not displayed in the plot). This comparison indicates significant recovery in naive Rlbp1+/+ mice and Rlbp1-/- mice receiving AAV8 vector compared to naive Rlbp1-/- mice at 2.5, 5, and 20 minutes postbleach (naive Rlbp1+/+: P ≤ 0.05, P ≤ 0.01, P ≤ 0.05; Rlbp1-/- + AAV8 vector. P ≤ 0.01, P ≤ 0.01, P ≤ 0.01 for the respective 2.5, 5, and 20 minute time points). No corresponding difference was detected at 0, 10, or 15 minutes (P > 0.05). No significant difference was detected between Rlbp1+/+ and Rlbp1-/- + AAV8 vector groups at any time point.

Mentions: We explored cone-driven ERG responses in Rlbp1+/+ and Rlbp1-/- mice to assess whether a measurable difference in function could be detected as described in a previous publication involving mice with a different mutation of Rlbp1.10 Like the mice in the previous publication, our Rlbp1-/- mice had cones that more slowly dark-adapted compared to Rlbp1+/+ controls, although the kinetics were different from what was described in the previously published strain. Photopic ERGs did not differ between our Rlbp1-/- and Rlbp1+/+ mice after overnight dark adaptation (data not shown). Immediately after bleaching with a 1200 cd (photopic) white LED light for one minute, photopic b-wave amplitudes of wild-type mice were reduced, but they recovered to 55–60% of the baseline value within 2.5–5 minutes (Figure 7a,d). In contrast, cone-mediated b-wave amplitudes from Rlbp1-/- mice did not detectably recover during the first 20 minutes after a bleach (Figure 7b,d). Ten weeks after subretinal injection of 1 × 109 vg of the self-complementary vector scAAV8-pRLBP1(short)-hRLBP1, Rlbp1-/- mice had rapid recovery kinetics similar to that observed in Rlbp1+/+ mice (Figure 7c,d), indicating that the vector restored rapid cone adaptation in Rlbp1-/- mice.


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)

scAAV8-pRLBP1(short)-hRLBP1 improves cone dark adaptation in Rlbp1-/- mice. (a–c) Cone-driven ERG-responses were extracted by the measurement of b-wave amplitudes from a paired-flash protocol at various time points following a 1 minute photobleach. Representative ERG traces from the paired-flash protocol at 0.0, 2.5, and 5.0 minutes post-bleach in Rlbp1+/+ mice, Rlbp1-/- mice, and Rlbp1-/- mice treated with a subretinal injection scAAV8-pRLBP1(short)-hRLBP1 10 weeks prior to recordings are shown (a–c respectively). (d) The graph displays average b-wave recoveries normalized to dark-adapted pre-bleach levels in Rlbp1+/+ (n = 4), Rlbp1-/- (n = 3), and vector treated Rlbp1-/- (n = 9) mice. The P values displayed in the plot compare the b-wave recovery at the 0 minute time point to later time points within the AAV8 vector-treated group (***P ≤ 0.001; ****P ≤ 0.0001). Analysis was also performed to compare recoveries between groups at each time point (not displayed in the plot). This comparison indicates significant recovery in naive Rlbp1+/+ mice and Rlbp1-/- mice receiving AAV8 vector compared to naive Rlbp1-/- mice at 2.5, 5, and 20 minutes postbleach (naive Rlbp1+/+: P ≤ 0.05, P ≤ 0.01, P ≤ 0.05; Rlbp1-/- + AAV8 vector. P ≤ 0.01, P ≤ 0.01, P ≤ 0.01 for the respective 2.5, 5, and 20 minute time points). No corresponding difference was detected at 0, 10, or 15 minutes (P > 0.05). No significant difference was detected between Rlbp1+/+ and Rlbp1-/- + AAV8 vector groups at any time point.
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fig7: scAAV8-pRLBP1(short)-hRLBP1 improves cone dark adaptation in Rlbp1-/- mice. (a–c) Cone-driven ERG-responses were extracted by the measurement of b-wave amplitudes from a paired-flash protocol at various time points following a 1 minute photobleach. Representative ERG traces from the paired-flash protocol at 0.0, 2.5, and 5.0 minutes post-bleach in Rlbp1+/+ mice, Rlbp1-/- mice, and Rlbp1-/- mice treated with a subretinal injection scAAV8-pRLBP1(short)-hRLBP1 10 weeks prior to recordings are shown (a–c respectively). (d) The graph displays average b-wave recoveries normalized to dark-adapted pre-bleach levels in Rlbp1+/+ (n = 4), Rlbp1-/- (n = 3), and vector treated Rlbp1-/- (n = 9) mice. The P values displayed in the plot compare the b-wave recovery at the 0 minute time point to later time points within the AAV8 vector-treated group (***P ≤ 0.001; ****P ≤ 0.0001). Analysis was also performed to compare recoveries between groups at each time point (not displayed in the plot). This comparison indicates significant recovery in naive Rlbp1+/+ mice and Rlbp1-/- mice receiving AAV8 vector compared to naive Rlbp1-/- mice at 2.5, 5, and 20 minutes postbleach (naive Rlbp1+/+: P ≤ 0.05, P ≤ 0.01, P ≤ 0.05; Rlbp1-/- + AAV8 vector. P ≤ 0.01, P ≤ 0.01, P ≤ 0.01 for the respective 2.5, 5, and 20 minute time points). No corresponding difference was detected at 0, 10, or 15 minutes (P > 0.05). No significant difference was detected between Rlbp1+/+ and Rlbp1-/- + AAV8 vector groups at any time point.
Mentions: We explored cone-driven ERG responses in Rlbp1+/+ and Rlbp1-/- mice to assess whether a measurable difference in function could be detected as described in a previous publication involving mice with a different mutation of Rlbp1.10 Like the mice in the previous publication, our Rlbp1-/- mice had cones that more slowly dark-adapted compared to Rlbp1+/+ controls, although the kinetics were different from what was described in the previously published strain. Photopic ERGs did not differ between our Rlbp1-/- and Rlbp1+/+ mice after overnight dark adaptation (data not shown). Immediately after bleaching with a 1200 cd (photopic) white LED light for one minute, photopic b-wave amplitudes of wild-type mice were reduced, but they recovered to 55–60% of the baseline value within 2.5–5 minutes (Figure 7a,d). In contrast, cone-mediated b-wave amplitudes from Rlbp1-/- mice did not detectably recover during the first 20 minutes after a bleach (Figure 7b,d). Ten weeks after subretinal injection of 1 × 109 vg of the self-complementary vector scAAV8-pRLBP1(short)-hRLBP1, Rlbp1-/- mice had rapid recovery kinetics similar to that observed in Rlbp1+/+ mice (Figure 7c,d), indicating that the vector restored rapid cone adaptation in Rlbp1-/- mice.

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