Limits...
Proof of concept for AAV2/5-mediated gene therapy in iPSC-derived retinal pigment epithelium of a choroideremia patient.

Cereso N, Pequignot MO, Robert L, Becker F, De Luca V, Nabholz N, Rigau V, De Vos J, Hamel CP, Kalatzis V - Mol Ther Methods Clin Dev (2014)

Bottom Line: We reprogrammed REP1-deficient fibroblasts from a CHM (-/y) patient into induced pluripotent stem cells (iPSCs), which we differentiated into retinal pigment epithelium (RPE).We assayed a panel of adeno-associated virus (AAV) vector serotypes and showed that AAV2/5 is the most efficient at transducing the iPSC-derived RPE and that CHM gene transfer normalizes the biochemical phenotype.We demonstrate the superiority of AAV2/5 in the human RPE and address the potential of patient iPSC-derived RPE to provide a proof-of-concept model for gene replacement in the absence of an appropriate animal model.

View Article: PubMed Central - PubMed

Affiliation: Inserm U1051, Institute for Neurosciences of Montpellier , Montpellier, France ; University of Montpellier 1 , Montpellier, France ; University of Montpellier 2 , Montpellier, France.

ABSTRACT
Inherited retinal dystrophies (IRDs) comprise a large group of genetically and clinically heterogeneous diseases that lead to progressive vision loss, for which a paucity of disease-mimicking animal models renders preclinical studies difficult. We sought to develop pertinent human cellular IRD models, beginning with choroideremia, caused by mutations in the CHM gene encoding Rab escort protein 1 (REP1). We reprogrammed REP1-deficient fibroblasts from a CHM (-/y) patient into induced pluripotent stem cells (iPSCs), which we differentiated into retinal pigment epithelium (RPE). This iPSC-derived RPE is a polarized monolayer with a classic morphology, expresses characteristic markers, is functional for fluid transport and phagocytosis, and mimics the biochemical phenotype of patients. We assayed a panel of adeno-associated virus (AAV) vector serotypes and showed that AAV2/5 is the most efficient at transducing the iPSC-derived RPE and that CHM gene transfer normalizes the biochemical phenotype. The high, and unmatched, in vitro transduction efficiency is likely aided by phagocytosis and mimics the scenario that an AAV vector encounters in vivo in the subretinal space. We demonstrate the superiority of AAV2/5 in the human RPE and address the potential of patient iPSC-derived RPE to provide a proof-of-concept model for gene replacement in the absence of an appropriate animal model.

No MeSH data available.


Related in: MedlinePlus

Characterization of the biochemical defect in CHM1 RPE. (a) A representative in vitro prenylation assay using a biotinylated prenyl donor followed by western blot analysis shows a weaker signal of incorporated biotin for the wild-type RPE than for the CHM1 RPE. (b) Semiquantification of the biotinylated Rab pool, after normalization with β-actin loading, confirms a significantly lower (*P < 0.05) relative quantity of biotinylated cytosolic Rabs in the wild-type RPE as opposed to that in CHM1 (data expressed as mean ± SEM, n = 3). (c) Following differential centrifugation and western blot analysis of Rab27A expression in the cytosolic (C) and membrane (M) fractions, a lower amount of Rab27A is detected in the cytosol of wild-type RPE, compared with that in the membrane fraction, for an equal β-actin loading. A less striking difference in Rab27A content between the two fractions is observed in CHM1 RPE. The depletion of the membrane fraction from the cytosol was controlled by hybridization with an anti-LAMP-2 antibody. (d) Semiquantification of the cytosolic fraction of Rab27A versus the total cellular content confirms a significantly lower quantity (*P < 0.05) in wild-type (WT) RPE, as opposed to the quantity in CHM1 RPE (data expressed as mean ± SEM, n = 3). LAMP, lysosomal membrane–associated protein; RPE, retinal pigment epithelium.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4362346&req=5

fig5: Characterization of the biochemical defect in CHM1 RPE. (a) A representative in vitro prenylation assay using a biotinylated prenyl donor followed by western blot analysis shows a weaker signal of incorporated biotin for the wild-type RPE than for the CHM1 RPE. (b) Semiquantification of the biotinylated Rab pool, after normalization with β-actin loading, confirms a significantly lower (*P < 0.05) relative quantity of biotinylated cytosolic Rabs in the wild-type RPE as opposed to that in CHM1 (data expressed as mean ± SEM, n = 3). (c) Following differential centrifugation and western blot analysis of Rab27A expression in the cytosolic (C) and membrane (M) fractions, a lower amount of Rab27A is detected in the cytosol of wild-type RPE, compared with that in the membrane fraction, for an equal β-actin loading. A less striking difference in Rab27A content between the two fractions is observed in CHM1 RPE. The depletion of the membrane fraction from the cytosol was controlled by hybridization with an anti-LAMP-2 antibody. (d) Semiquantification of the cytosolic fraction of Rab27A versus the total cellular content confirms a significantly lower quantity (*P < 0.05) in wild-type (WT) RPE, as opposed to the quantity in CHM1 RPE (data expressed as mean ± SEM, n = 3). LAMP, lysosomal membrane–associated protein; RPE, retinal pigment epithelium.

Mentions: In order to determine whether the iPSC-derived RPE from the individual CHM1 reproduced the biochemical defect of patients, we used two different techniques to assay the prenylation status of intracellular Rabs, a reflection of the activity of REP1. First, we assayed the size of the unprenylated Rab pool in the cells using an in vitro prenylation assay. To this end, we added recombinant Rab geranylgeranyl transferase, REP1, and the biotinylated prenyl donor to the lysate of wild-type and CHM1 cells. Thus, if an unprenylated Rab pool were available for prenylation, the integrated biotin could be detected by western blot analysis using horseradish peroxidase–conjugated streptavidin (Figure 5a). The semiquantification of three experiments (Figure 5b) showed that a significantly lower level (P < 0.05) of biotinylated Rab proteins was detected in the wild-type RPE than in the CHM1 RPE, consistent with the fact that, in the presence of REP1 and REP2, most Rabs are prenylated and membrane bound. Second, we specifically assayed the subcellular distribution of Rab27A, a Rab protein highly expressed in the retina.28 By differential centrifugation, we separated the cytosolic and membrane fractions of wild-type and CHM1 cell lysates and analyzed the respective contents of Rab27A by western blot analysis with a specific antibody (Figure 5c). The depletion of the membrane fraction from the cytosol was verified by hybridization with an antibody against the lysosomal membrane–associated protein LAMP-2. The semiquantification of three experiments (Figure 5d) showed that the cytosolic versus membrane-bound Rab27A content was significantly lower (P < 0.05) in wild-type RPE as compared with that in CHM1 RPE.


Proof of concept for AAV2/5-mediated gene therapy in iPSC-derived retinal pigment epithelium of a choroideremia patient.

Cereso N, Pequignot MO, Robert L, Becker F, De Luca V, Nabholz N, Rigau V, De Vos J, Hamel CP, Kalatzis V - Mol Ther Methods Clin Dev (2014)

Characterization of the biochemical defect in CHM1 RPE. (a) A representative in vitro prenylation assay using a biotinylated prenyl donor followed by western blot analysis shows a weaker signal of incorporated biotin for the wild-type RPE than for the CHM1 RPE. (b) Semiquantification of the biotinylated Rab pool, after normalization with β-actin loading, confirms a significantly lower (*P < 0.05) relative quantity of biotinylated cytosolic Rabs in the wild-type RPE as opposed to that in CHM1 (data expressed as mean ± SEM, n = 3). (c) Following differential centrifugation and western blot analysis of Rab27A expression in the cytosolic (C) and membrane (M) fractions, a lower amount of Rab27A is detected in the cytosol of wild-type RPE, compared with that in the membrane fraction, for an equal β-actin loading. A less striking difference in Rab27A content between the two fractions is observed in CHM1 RPE. The depletion of the membrane fraction from the cytosol was controlled by hybridization with an anti-LAMP-2 antibody. (d) Semiquantification of the cytosolic fraction of Rab27A versus the total cellular content confirms a significantly lower quantity (*P < 0.05) in wild-type (WT) RPE, as opposed to the quantity in CHM1 RPE (data expressed as mean ± SEM, n = 3). LAMP, lysosomal membrane–associated protein; RPE, retinal pigment epithelium.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362346&req=5

fig5: Characterization of the biochemical defect in CHM1 RPE. (a) A representative in vitro prenylation assay using a biotinylated prenyl donor followed by western blot analysis shows a weaker signal of incorporated biotin for the wild-type RPE than for the CHM1 RPE. (b) Semiquantification of the biotinylated Rab pool, after normalization with β-actin loading, confirms a significantly lower (*P < 0.05) relative quantity of biotinylated cytosolic Rabs in the wild-type RPE as opposed to that in CHM1 (data expressed as mean ± SEM, n = 3). (c) Following differential centrifugation and western blot analysis of Rab27A expression in the cytosolic (C) and membrane (M) fractions, a lower amount of Rab27A is detected in the cytosol of wild-type RPE, compared with that in the membrane fraction, for an equal β-actin loading. A less striking difference in Rab27A content between the two fractions is observed in CHM1 RPE. The depletion of the membrane fraction from the cytosol was controlled by hybridization with an anti-LAMP-2 antibody. (d) Semiquantification of the cytosolic fraction of Rab27A versus the total cellular content confirms a significantly lower quantity (*P < 0.05) in wild-type (WT) RPE, as opposed to the quantity in CHM1 RPE (data expressed as mean ± SEM, n = 3). LAMP, lysosomal membrane–associated protein; RPE, retinal pigment epithelium.
Mentions: In order to determine whether the iPSC-derived RPE from the individual CHM1 reproduced the biochemical defect of patients, we used two different techniques to assay the prenylation status of intracellular Rabs, a reflection of the activity of REP1. First, we assayed the size of the unprenylated Rab pool in the cells using an in vitro prenylation assay. To this end, we added recombinant Rab geranylgeranyl transferase, REP1, and the biotinylated prenyl donor to the lysate of wild-type and CHM1 cells. Thus, if an unprenylated Rab pool were available for prenylation, the integrated biotin could be detected by western blot analysis using horseradish peroxidase–conjugated streptavidin (Figure 5a). The semiquantification of three experiments (Figure 5b) showed that a significantly lower level (P < 0.05) of biotinylated Rab proteins was detected in the wild-type RPE than in the CHM1 RPE, consistent with the fact that, in the presence of REP1 and REP2, most Rabs are prenylated and membrane bound. Second, we specifically assayed the subcellular distribution of Rab27A, a Rab protein highly expressed in the retina.28 By differential centrifugation, we separated the cytosolic and membrane fractions of wild-type and CHM1 cell lysates and analyzed the respective contents of Rab27A by western blot analysis with a specific antibody (Figure 5c). The depletion of the membrane fraction from the cytosol was verified by hybridization with an antibody against the lysosomal membrane–associated protein LAMP-2. The semiquantification of three experiments (Figure 5d) showed that the cytosolic versus membrane-bound Rab27A content was significantly lower (P < 0.05) in wild-type RPE as compared with that in CHM1 RPE.

Bottom Line: We reprogrammed REP1-deficient fibroblasts from a CHM (-/y) patient into induced pluripotent stem cells (iPSCs), which we differentiated into retinal pigment epithelium (RPE).We assayed a panel of adeno-associated virus (AAV) vector serotypes and showed that AAV2/5 is the most efficient at transducing the iPSC-derived RPE and that CHM gene transfer normalizes the biochemical phenotype.We demonstrate the superiority of AAV2/5 in the human RPE and address the potential of patient iPSC-derived RPE to provide a proof-of-concept model for gene replacement in the absence of an appropriate animal model.

View Article: PubMed Central - PubMed

Affiliation: Inserm U1051, Institute for Neurosciences of Montpellier , Montpellier, France ; University of Montpellier 1 , Montpellier, France ; University of Montpellier 2 , Montpellier, France.

ABSTRACT
Inherited retinal dystrophies (IRDs) comprise a large group of genetically and clinically heterogeneous diseases that lead to progressive vision loss, for which a paucity of disease-mimicking animal models renders preclinical studies difficult. We sought to develop pertinent human cellular IRD models, beginning with choroideremia, caused by mutations in the CHM gene encoding Rab escort protein 1 (REP1). We reprogrammed REP1-deficient fibroblasts from a CHM (-/y) patient into induced pluripotent stem cells (iPSCs), which we differentiated into retinal pigment epithelium (RPE). This iPSC-derived RPE is a polarized monolayer with a classic morphology, expresses characteristic markers, is functional for fluid transport and phagocytosis, and mimics the biochemical phenotype of patients. We assayed a panel of adeno-associated virus (AAV) vector serotypes and showed that AAV2/5 is the most efficient at transducing the iPSC-derived RPE and that CHM gene transfer normalizes the biochemical phenotype. The high, and unmatched, in vitro transduction efficiency is likely aided by phagocytosis and mimics the scenario that an AAV vector encounters in vivo in the subretinal space. We demonstrate the superiority of AAV2/5 in the human RPE and address the potential of patient iPSC-derived RPE to provide a proof-of-concept model for gene replacement in the absence of an appropriate animal model.

No MeSH data available.


Related in: MedlinePlus