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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 expression levels of the human RLBP1 transgene under the transcriptional control of different promoters. (a,b) Relative mRNA levels of vector-mediated RLBP1 expression were assayed using qPCR probes specific for human RLBP1 or mouse Rlbp1 and RNA extracted from wild-type mouse neural retina or eye cups 4 weeks after subretinal injection with rAAV vectors carrying different promoters or genome conformations driving hRLBP1 expression. Relative expression of RLBP1 is graphed as a mean fold change from WT mouse Rlbp1 expression +SEM. Naive (uninjected) neural retina (nr) and eye cup (ec) samples were also included at each dose (n = 12). (a) Relative RLBP1 expression mediated by a dose of 1 × 109 vg/eye of vectors ssAAV8-pRLBP1(long)-hRLBP1 (nr n = 20; ec n = 17), ssAAV8-pRLBP1(short)-hRLBP1 (nr+ec n = 8), scAAV8-pRLBP1(short)-hRLBP1 (nr n = 19; ec n = 16), or ssAAV8-pRPE65-hRLBP1 (nr n = 19; ec n = 15). (b) Relative RLBP1 expression mediated by a dose of 1 × 108 vg/eye of vectors ssAAV8-pRLBP1(long)-hRLBP1 (nr n = 9; ec n = 7), ssAAV8-pRLBP1(short)-hRLBP1 (nr+ec n = 4), scAAV8-pRLBP1(short)-hRLBP1 (nr+ec n = 9), or ssAAV8-pRPE65-hRLBP1 (nr+ec n = 8). WT, wild-type; ss, single-stranded vector genome; sc, self-complementary vector genome. Calculations of P values compared expression levels to naive wild-type eyes; **P ≤ 0.01; ****P ≤ 0.0001.
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fig2: Vector mediated expression levels of the human RLBP1 transgene under the transcriptional control of different promoters. (a,b) Relative mRNA levels of vector-mediated RLBP1 expression were assayed using qPCR probes specific for human RLBP1 or mouse Rlbp1 and RNA extracted from wild-type mouse neural retina or eye cups 4 weeks after subretinal injection with rAAV vectors carrying different promoters or genome conformations driving hRLBP1 expression. Relative expression of RLBP1 is graphed as a mean fold change from WT mouse Rlbp1 expression +SEM. Naive (uninjected) neural retina (nr) and eye cup (ec) samples were also included at each dose (n = 12). (a) Relative RLBP1 expression mediated by a dose of 1 × 109 vg/eye of vectors ssAAV8-pRLBP1(long)-hRLBP1 (nr n = 20; ec n = 17), ssAAV8-pRLBP1(short)-hRLBP1 (nr+ec n = 8), scAAV8-pRLBP1(short)-hRLBP1 (nr n = 19; ec n = 16), or ssAAV8-pRPE65-hRLBP1 (nr n = 19; ec n = 15). (b) Relative RLBP1 expression mediated by a dose of 1 × 108 vg/eye of vectors ssAAV8-pRLBP1(long)-hRLBP1 (nr n = 9; ec n = 7), ssAAV8-pRLBP1(short)-hRLBP1 (nr+ec n = 4), scAAV8-pRLBP1(short)-hRLBP1 (nr+ec n = 9), or ssAAV8-pRPE65-hRLBP1 (nr+ec n = 8). WT, wild-type; ss, single-stranded vector genome; sc, self-complementary vector genome. Calculations of P values compared expression levels to naive wild-type eyes; **P ≤ 0.01; ****P ≤ 0.0001.

Mentions: Three of the promoters were further characterized in mice by evaluating four rAAV8 vectors in which the promoters drove the expression of a cDNA sequence derived from the human RLBP1 gene: one with the pRLBP1(short) promoter in a single-stranded genome called ssAAV8-pRLBP1(short)-hRLBP1, one with the pRLBP1(short) promoter in a self-complementary genome called scAAV8-pRLBP1(short)-hRLBP1, one with the pRLBP1(long) promoter in a single-stranded genome called ssAAV8-pRLBP1(long)-hRLBP1, and one with the pRPE65 promoter in a single-stranded genome called ssAAV8-pRPE65-hRLBP1. Levels of human RLBP1 (hRLBP1) mRNA were measured in the neural retina and the eye cup (which included the whole eye minus the lens and neural retina) four weeks after subretinal injections of 1 × 108 or 1 × 109 vg/eye in wild-type mice. At a dose of 1 × 109 vg/eye (Figure 2a) of the self-complementary vector with the short RLBP1 promoter, scAAV8-pRLBP1(short)-hRLBP1, had the highest expression in the neural retina and the eye cup. At a 10-fold lower dose of 1 × 108 vg/eye (Figure 2b), the only vector that showed substantial expression levels of the hRLBP1 transgene in neural retina was scAAV8-pRLBP1(short)-hRLBP1. This vector also produced the highest level of hRLBP1 transgene expression in the eye cup at a dose of 1 × 108 vg/eye. Due to its higher transduction efficiency and cell-specific expression pattern, this self-complementary rAAV8 vector with the short RLBP1 promoter (scAAV8-pRLBP1(short)-hRLBP1) was chosen for subsequent efficacy studies in 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)

Vector mediated expression levels of the human RLBP1 transgene under the transcriptional control of different promoters. (a,b) Relative mRNA levels of vector-mediated RLBP1 expression were assayed using qPCR probes specific for human RLBP1 or mouse Rlbp1 and RNA extracted from wild-type mouse neural retina or eye cups 4 weeks after subretinal injection with rAAV vectors carrying different promoters or genome conformations driving hRLBP1 expression. Relative expression of RLBP1 is graphed as a mean fold change from WT mouse Rlbp1 expression +SEM. Naive (uninjected) neural retina (nr) and eye cup (ec) samples were also included at each dose (n = 12). (a) Relative RLBP1 expression mediated by a dose of 1 × 109 vg/eye of vectors ssAAV8-pRLBP1(long)-hRLBP1 (nr n = 20; ec n = 17), ssAAV8-pRLBP1(short)-hRLBP1 (nr+ec n = 8), scAAV8-pRLBP1(short)-hRLBP1 (nr n = 19; ec n = 16), or ssAAV8-pRPE65-hRLBP1 (nr n = 19; ec n = 15). (b) Relative RLBP1 expression mediated by a dose of 1 × 108 vg/eye of vectors ssAAV8-pRLBP1(long)-hRLBP1 (nr n = 9; ec n = 7), ssAAV8-pRLBP1(short)-hRLBP1 (nr+ec n = 4), scAAV8-pRLBP1(short)-hRLBP1 (nr+ec n = 9), or ssAAV8-pRPE65-hRLBP1 (nr+ec n = 8). WT, wild-type; ss, single-stranded vector genome; sc, self-complementary vector genome. Calculations of P values compared expression levels to naive wild-type eyes; **P ≤ 0.01; ****P ≤ 0.0001.
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fig2: Vector mediated expression levels of the human RLBP1 transgene under the transcriptional control of different promoters. (a,b) Relative mRNA levels of vector-mediated RLBP1 expression were assayed using qPCR probes specific for human RLBP1 or mouse Rlbp1 and RNA extracted from wild-type mouse neural retina or eye cups 4 weeks after subretinal injection with rAAV vectors carrying different promoters or genome conformations driving hRLBP1 expression. Relative expression of RLBP1 is graphed as a mean fold change from WT mouse Rlbp1 expression +SEM. Naive (uninjected) neural retina (nr) and eye cup (ec) samples were also included at each dose (n = 12). (a) Relative RLBP1 expression mediated by a dose of 1 × 109 vg/eye of vectors ssAAV8-pRLBP1(long)-hRLBP1 (nr n = 20; ec n = 17), ssAAV8-pRLBP1(short)-hRLBP1 (nr+ec n = 8), scAAV8-pRLBP1(short)-hRLBP1 (nr n = 19; ec n = 16), or ssAAV8-pRPE65-hRLBP1 (nr n = 19; ec n = 15). (b) Relative RLBP1 expression mediated by a dose of 1 × 108 vg/eye of vectors ssAAV8-pRLBP1(long)-hRLBP1 (nr n = 9; ec n = 7), ssAAV8-pRLBP1(short)-hRLBP1 (nr+ec n = 4), scAAV8-pRLBP1(short)-hRLBP1 (nr+ec n = 9), or ssAAV8-pRPE65-hRLBP1 (nr+ec n = 8). WT, wild-type; ss, single-stranded vector genome; sc, self-complementary vector genome. Calculations of P values compared expression levels to naive wild-type eyes; **P ≤ 0.01; ****P ≤ 0.0001.
Mentions: Three of the promoters were further characterized in mice by evaluating four rAAV8 vectors in which the promoters drove the expression of a cDNA sequence derived from the human RLBP1 gene: one with the pRLBP1(short) promoter in a single-stranded genome called ssAAV8-pRLBP1(short)-hRLBP1, one with the pRLBP1(short) promoter in a self-complementary genome called scAAV8-pRLBP1(short)-hRLBP1, one with the pRLBP1(long) promoter in a single-stranded genome called ssAAV8-pRLBP1(long)-hRLBP1, and one with the pRPE65 promoter in a single-stranded genome called ssAAV8-pRPE65-hRLBP1. Levels of human RLBP1 (hRLBP1) mRNA were measured in the neural retina and the eye cup (which included the whole eye minus the lens and neural retina) four weeks after subretinal injections of 1 × 108 or 1 × 109 vg/eye in wild-type mice. At a dose of 1 × 109 vg/eye (Figure 2a) of the self-complementary vector with the short RLBP1 promoter, scAAV8-pRLBP1(short)-hRLBP1, had the highest expression in the neural retina and the eye cup. At a 10-fold lower dose of 1 × 108 vg/eye (Figure 2b), the only vector that showed substantial expression levels of the hRLBP1 transgene in neural retina was scAAV8-pRLBP1(short)-hRLBP1. This vector also produced the highest level of hRLBP1 transgene expression in the eye cup at a dose of 1 × 108 vg/eye. Due to its higher transduction efficiency and cell-specific expression pattern, this self-complementary rAAV8 vector with the short RLBP1 promoter (scAAV8-pRLBP1(short)-hRLBP1) was chosen for subsequent efficacy studies in 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