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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

Characterization of the Rlbp1-/- mouse. (a) The horizontal bar is a schematic representation of the Rlbp1 targeted deletion in the Lexicon TF0133 mouse line. Primers used for genotyping are depicted as arrows (P-9, P-3, and P-14). (b) PCR analysis of DNA from Rlbp1-/-, Rlbp1+/-, and Rlbp1+/+ mice using primers P-9, P-3, and P-14 show the expected 854 bp amplicon of the WT allele and the 358 bp amplicon of the allele carrying the deletion. (c,d) Paraffin sections of Rlbp1+/+ (c) and Rlbp1-/- (d) mouse eyes stained with an antibody against CRALBP. (e,f) Hematoxylin and eosin stained paraffin sections of 16-month-old Rlbp1+/+ (e) and Rlbp1-/- (f) mouse eyes. (g) Lysates of neural retina from uninjected Rlbp1-/- (KO uninj.), Rlbp1-/- injected with 1 × 109 vg/eye of scAAV8-pRLBP1(short)-hRLBP1 (KO, vector inj.), and uninjected Rlbp1+/+ (WT uninj.) mice were analyzed by western blot using a primary antibody against CRALBP. Arrow, end feet of Müller cells; arrow head, Müller cell processes; diamond, cell bodies of inner nuclear layer; asterisk, RPE; scale bars, 50 µm in c+d and 100 µm in e+f.
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fig3: Characterization of the Rlbp1-/- mouse. (a) The horizontal bar is a schematic representation of the Rlbp1 targeted deletion in the Lexicon TF0133 mouse line. Primers used for genotyping are depicted as arrows (P-9, P-3, and P-14). (b) PCR analysis of DNA from Rlbp1-/-, Rlbp1+/-, and Rlbp1+/+ mice using primers P-9, P-3, and P-14 show the expected 854 bp amplicon of the WT allele and the 358 bp amplicon of the allele carrying the deletion. (c,d) Paraffin sections of Rlbp1+/+ (c) and Rlbp1-/- (d) mouse eyes stained with an antibody against CRALBP. (e,f) Hematoxylin and eosin stained paraffin sections of 16-month-old Rlbp1+/+ (e) and Rlbp1-/- (f) mouse eyes. (g) Lysates of neural retina from uninjected Rlbp1-/- (KO uninj.), Rlbp1-/- injected with 1 × 109 vg/eye of scAAV8-pRLBP1(short)-hRLBP1 (KO, vector inj.), and uninjected Rlbp1+/+ (WT uninj.) mice were analyzed by western blot using a primary antibody against CRALBP. Arrow, end feet of Müller cells; arrow head, Müller cell processes; diamond, cell bodies of inner nuclear layer; asterisk, RPE; scale bars, 50 µm in c+d and 100 µm in e+f.

Mentions: The Lexicon mouse line TF0133 used in this research has a 496-bp deletion that begins in exon 3 and extends to the end of exon 4. The deletion removes the initiation methionine codon in exon 3 (Figure 3a). PCR using primers that straddle the deletion breakpoints amplified fragments from genomic DNA of Rlbp1+/+, Rlbp1+/-, and Rlbp1-/- mice that corresponded in length to the presence or absence of the deletion (Figure 3b). The mutation creates a allele, as confirmed through analysis of CRALBP protein. Specifically, the rabbit polyclonal anti-CRALBP antibody (15356-1-AP) detected CRALBP in wild-type mice in both the RPE and in the neural retina. The pattern of staining in the neural retina indicated that CRALBP was in Müller cell bodies, processes, and end feet (Figure 3c). No CRALBP was detected in the neural retina or the RPE in Rlbp1-/- mice (Figure 3d). The thickness of the retina of Rlbp1+/+ mice and Rlbp1-/- mice was measured at 4, 10, and 16 months of age. There was no detectable reduction in retinal thickness up to 16 months of age (Figure 3e,f).


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)

Characterization of the Rlbp1-/- mouse. (a) The horizontal bar is a schematic representation of the Rlbp1 targeted deletion in the Lexicon TF0133 mouse line. Primers used for genotyping are depicted as arrows (P-9, P-3, and P-14). (b) PCR analysis of DNA from Rlbp1-/-, Rlbp1+/-, and Rlbp1+/+ mice using primers P-9, P-3, and P-14 show the expected 854 bp amplicon of the WT allele and the 358 bp amplicon of the allele carrying the deletion. (c,d) Paraffin sections of Rlbp1+/+ (c) and Rlbp1-/- (d) mouse eyes stained with an antibody against CRALBP. (e,f) Hematoxylin and eosin stained paraffin sections of 16-month-old Rlbp1+/+ (e) and Rlbp1-/- (f) mouse eyes. (g) Lysates of neural retina from uninjected Rlbp1-/- (KO uninj.), Rlbp1-/- injected with 1 × 109 vg/eye of scAAV8-pRLBP1(short)-hRLBP1 (KO, vector inj.), and uninjected Rlbp1+/+ (WT uninj.) mice were analyzed by western blot using a primary antibody against CRALBP. Arrow, end feet of Müller cells; arrow head, Müller cell processes; diamond, cell bodies of inner nuclear layer; asterisk, RPE; scale bars, 50 µm in c+d and 100 µm in e+f.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Characterization of the Rlbp1-/- mouse. (a) The horizontal bar is a schematic representation of the Rlbp1 targeted deletion in the Lexicon TF0133 mouse line. Primers used for genotyping are depicted as arrows (P-9, P-3, and P-14). (b) PCR analysis of DNA from Rlbp1-/-, Rlbp1+/-, and Rlbp1+/+ mice using primers P-9, P-3, and P-14 show the expected 854 bp amplicon of the WT allele and the 358 bp amplicon of the allele carrying the deletion. (c,d) Paraffin sections of Rlbp1+/+ (c) and Rlbp1-/- (d) mouse eyes stained with an antibody against CRALBP. (e,f) Hematoxylin and eosin stained paraffin sections of 16-month-old Rlbp1+/+ (e) and Rlbp1-/- (f) mouse eyes. (g) Lysates of neural retina from uninjected Rlbp1-/- (KO uninj.), Rlbp1-/- injected with 1 × 109 vg/eye of scAAV8-pRLBP1(short)-hRLBP1 (KO, vector inj.), and uninjected Rlbp1+/+ (WT uninj.) mice were analyzed by western blot using a primary antibody against CRALBP. Arrow, end feet of Müller cells; arrow head, Müller cell processes; diamond, cell bodies of inner nuclear layer; asterisk, RPE; scale bars, 50 µm in c+d and 100 µm in e+f.
Mentions: The Lexicon mouse line TF0133 used in this research has a 496-bp deletion that begins in exon 3 and extends to the end of exon 4. The deletion removes the initiation methionine codon in exon 3 (Figure 3a). PCR using primers that straddle the deletion breakpoints amplified fragments from genomic DNA of Rlbp1+/+, Rlbp1+/-, and Rlbp1-/- mice that corresponded in length to the presence or absence of the deletion (Figure 3b). The mutation creates a allele, as confirmed through analysis of CRALBP protein. Specifically, the rabbit polyclonal anti-CRALBP antibody (15356-1-AP) detected CRALBP in wild-type mice in both the RPE and in the neural retina. The pattern of staining in the neural retina indicated that CRALBP was in Müller cell bodies, processes, and end feet (Figure 3c). No CRALBP was detected in the neural retina or the RPE in Rlbp1-/- mice (Figure 3d). The thickness of the retina of Rlbp1+/+ mice and Rlbp1-/- mice was measured at 4, 10, and 16 months of age. There was no detectable reduction in retinal thickness up to 16 months of age (Figure 3e,f).

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