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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

Generation and characterization of iPSC-derived RPE. (a) Pigmented foci in confluent iPSC plates following bFGF depletion (arrowheads). (b) At confluence, passaged pigmented foci form a layer of polygonal pigmented cells. Bar = 100 µm. (c) Semi-thin section of iPSC-derived epithelium cultured on a porous filter and stained with toluidine blue demonstrates a regular monolayer (in blue) superposed on the filter (in white). Bar = 50 µm. (d) Transmission electron microscopy shows the iPSC-derived monolayer as a polarized epithelium with microvilli (m) on the apical side, desmosomes (arrowheads) at the apical junctions, melanosomes (asterisks) distributed throughout the cytosol, a nucleus (n) on the basal side, and a basal lamina (arrow) between the epithelium and the filter. Bar = 2 µm. (e) Expression of classic RPE genes, as determined by reverse transcriptase (RT)–polymerase chain reaction analysis in both wild-type (WT) and patient (CHM1) RPEs in the presence of RT (+RT). In the absence of RT (−RT) or complementary DNA (cDNA) (−), an amplicon was not detected. Immunofluorescence studies of the RPE monolayer, followed by confocal analysis, demonstrate the expression of (f) MERTK in the microvilli (in red), (g) CRALBP and (h) RPE65 in the cytoplasm (in green), and (i) ZO-1 at the apical junctions (in red). Bars = 100 µm (in f, g), 30 µm (in h), and 50 µm (in i). Apicobasal fluid transport causes the RPE to form fluid-filled domes, detaching it from the cell culture plate: (j) focus on the RPE adhered to the plate; (k) focus on the RPE at the top of the dome. Bars = 100 µm. (l) Confocal analysis of the RPE 6 hours postincubation with FluoSpheres showing the internalized beads (in green), nuclei (in blue), and F-actin (in red). Bar = 15 µm. (m) Flow cytometry analysis showing the percentage of RPE cells that internalized FluoSpheres over time. bFGF, basic fibroblast growth factor; BEST1, bestrophin 1; CRALPB, cellular retinaldehyde-binding protein; GAPDH, glycerglyceraldehyde 3-phosphate dehydrogenase; iPSC, induced pluripotent stem cell; LRAT, lecithin retinol acyltransferase; MERTK , C-mer proto-oncogene tyrosine kinase; PAX6 , paired box 6; RDH, retinal dehydrogenase 5; RLBP1, retinaldehyde binding protein 1; RPE, retinal pigment epithelium; TYR, tyrosinase; ZO1, zona occludens protein 1.
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fig4: Generation and characterization of iPSC-derived RPE. (a) Pigmented foci in confluent iPSC plates following bFGF depletion (arrowheads). (b) At confluence, passaged pigmented foci form a layer of polygonal pigmented cells. Bar = 100 µm. (c) Semi-thin section of iPSC-derived epithelium cultured on a porous filter and stained with toluidine blue demonstrates a regular monolayer (in blue) superposed on the filter (in white). Bar = 50 µm. (d) Transmission electron microscopy shows the iPSC-derived monolayer as a polarized epithelium with microvilli (m) on the apical side, desmosomes (arrowheads) at the apical junctions, melanosomes (asterisks) distributed throughout the cytosol, a nucleus (n) on the basal side, and a basal lamina (arrow) between the epithelium and the filter. Bar = 2 µm. (e) Expression of classic RPE genes, as determined by reverse transcriptase (RT)–polymerase chain reaction analysis in both wild-type (WT) and patient (CHM1) RPEs in the presence of RT (+RT). In the absence of RT (−RT) or complementary DNA (cDNA) (−), an amplicon was not detected. Immunofluorescence studies of the RPE monolayer, followed by confocal analysis, demonstrate the expression of (f) MERTK in the microvilli (in red), (g) CRALBP and (h) RPE65 in the cytoplasm (in green), and (i) ZO-1 at the apical junctions (in red). Bars = 100 µm (in f, g), 30 µm (in h), and 50 µm (in i). Apicobasal fluid transport causes the RPE to form fluid-filled domes, detaching it from the cell culture plate: (j) focus on the RPE adhered to the plate; (k) focus on the RPE at the top of the dome. Bars = 100 µm. (l) Confocal analysis of the RPE 6 hours postincubation with FluoSpheres showing the internalized beads (in green), nuclei (in blue), and F-actin (in red). Bar = 15 µm. (m) Flow cytometry analysis showing the percentage of RPE cells that internalized FluoSpheres over time. bFGF, basic fibroblast growth factor; BEST1, bestrophin 1; CRALPB, cellular retinaldehyde-binding protein; GAPDH, glycerglyceraldehyde 3-phosphate dehydrogenase; iPSC, induced pluripotent stem cell; LRAT, lecithin retinol acyltransferase; MERTK , C-mer proto-oncogene tyrosine kinase; PAX6 , paired box 6; RDH, retinal dehydrogenase 5; RLBP1, retinaldehyde binding protein 1; RPE, retinal pigment epithelium; TYR, tyrosinase; ZO1, zona occludens protein 1.

Mentions: We used a spontaneous differentiation protocol to generate RPE from wild-type M4C7 and CHM1 iPSCs. Approximately 15 days after the iPSCs were cultured to confluence and the basic fibroblast growth factors were removed from the media, pigmented foci appeared in the plates (Figure 4a). These foci were mechanically passaged, and at confluence, from passage (P) 2, gave rise to a homogeneous layer of polygonal pigmented cells (Figure 4b) characteristic of RPE. Seeding the cells on translucent porous filters allowed sectioning and histological analysis. Observation of semi-thin sections demonstrated that the cell layer was a regular monolayer (Figure 4c). Transmission electron microscopy showed that the monolayer was a polarized epithelium with microvilli on the apical side, a nucleus on the basal side, and cytosolic melanosomes and desmosomes indicative of tight junctions (Figure 4d). The epithelium appeared to secrete a basal lamina detectable between the RPE cells and the filter. Reverse transcriptase–PCR (RT-PCR) studies (Figure 4e) demonstrated that the iPSC-derived epithelium expressed classic genes for the visual cycle (such as RLBP1, RPE65, LRAT, and RDH5), retinal development (PAX6), phagocytosis (MERTK), pigmentation (TYR), ion transport (BEST1), and cell adhesion (ZO-1). Furthermore, IF studies showed that MERTK was localized in the apical microvilli (Figure 4f), CRALBP (Figure 4g) and RPE65 (Figure 4h) in the cytoplasm, and ZO-1 at the apical junctions (Figure 4i), in accordance with their respective roles. Moreover, the presence of desmosomes was consistent with the positive ZO-1-labeling.


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)

Generation and characterization of iPSC-derived RPE. (a) Pigmented foci in confluent iPSC plates following bFGF depletion (arrowheads). (b) At confluence, passaged pigmented foci form a layer of polygonal pigmented cells. Bar = 100 µm. (c) Semi-thin section of iPSC-derived epithelium cultured on a porous filter and stained with toluidine blue demonstrates a regular monolayer (in blue) superposed on the filter (in white). Bar = 50 µm. (d) Transmission electron microscopy shows the iPSC-derived monolayer as a polarized epithelium with microvilli (m) on the apical side, desmosomes (arrowheads) at the apical junctions, melanosomes (asterisks) distributed throughout the cytosol, a nucleus (n) on the basal side, and a basal lamina (arrow) between the epithelium and the filter. Bar = 2 µm. (e) Expression of classic RPE genes, as determined by reverse transcriptase (RT)–polymerase chain reaction analysis in both wild-type (WT) and patient (CHM1) RPEs in the presence of RT (+RT). In the absence of RT (−RT) or complementary DNA (cDNA) (−), an amplicon was not detected. Immunofluorescence studies of the RPE monolayer, followed by confocal analysis, demonstrate the expression of (f) MERTK in the microvilli (in red), (g) CRALBP and (h) RPE65 in the cytoplasm (in green), and (i) ZO-1 at the apical junctions (in red). Bars = 100 µm (in f, g), 30 µm (in h), and 50 µm (in i). Apicobasal fluid transport causes the RPE to form fluid-filled domes, detaching it from the cell culture plate: (j) focus on the RPE adhered to the plate; (k) focus on the RPE at the top of the dome. Bars = 100 µm. (l) Confocal analysis of the RPE 6 hours postincubation with FluoSpheres showing the internalized beads (in green), nuclei (in blue), and F-actin (in red). Bar = 15 µm. (m) Flow cytometry analysis showing the percentage of RPE cells that internalized FluoSpheres over time. bFGF, basic fibroblast growth factor; BEST1, bestrophin 1; CRALPB, cellular retinaldehyde-binding protein; GAPDH, glycerglyceraldehyde 3-phosphate dehydrogenase; iPSC, induced pluripotent stem cell; LRAT, lecithin retinol acyltransferase; MERTK , C-mer proto-oncogene tyrosine kinase; PAX6 , paired box 6; RDH, retinal dehydrogenase 5; RLBP1, retinaldehyde binding protein 1; RPE, retinal pigment epithelium; TYR, tyrosinase; ZO1, zona occludens protein 1.
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fig4: Generation and characterization of iPSC-derived RPE. (a) Pigmented foci in confluent iPSC plates following bFGF depletion (arrowheads). (b) At confluence, passaged pigmented foci form a layer of polygonal pigmented cells. Bar = 100 µm. (c) Semi-thin section of iPSC-derived epithelium cultured on a porous filter and stained with toluidine blue demonstrates a regular monolayer (in blue) superposed on the filter (in white). Bar = 50 µm. (d) Transmission electron microscopy shows the iPSC-derived monolayer as a polarized epithelium with microvilli (m) on the apical side, desmosomes (arrowheads) at the apical junctions, melanosomes (asterisks) distributed throughout the cytosol, a nucleus (n) on the basal side, and a basal lamina (arrow) between the epithelium and the filter. Bar = 2 µm. (e) Expression of classic RPE genes, as determined by reverse transcriptase (RT)–polymerase chain reaction analysis in both wild-type (WT) and patient (CHM1) RPEs in the presence of RT (+RT). In the absence of RT (−RT) or complementary DNA (cDNA) (−), an amplicon was not detected. Immunofluorescence studies of the RPE monolayer, followed by confocal analysis, demonstrate the expression of (f) MERTK in the microvilli (in red), (g) CRALBP and (h) RPE65 in the cytoplasm (in green), and (i) ZO-1 at the apical junctions (in red). Bars = 100 µm (in f, g), 30 µm (in h), and 50 µm (in i). Apicobasal fluid transport causes the RPE to form fluid-filled domes, detaching it from the cell culture plate: (j) focus on the RPE adhered to the plate; (k) focus on the RPE at the top of the dome. Bars = 100 µm. (l) Confocal analysis of the RPE 6 hours postincubation with FluoSpheres showing the internalized beads (in green), nuclei (in blue), and F-actin (in red). Bar = 15 µm. (m) Flow cytometry analysis showing the percentage of RPE cells that internalized FluoSpheres over time. bFGF, basic fibroblast growth factor; BEST1, bestrophin 1; CRALPB, cellular retinaldehyde-binding protein; GAPDH, glycerglyceraldehyde 3-phosphate dehydrogenase; iPSC, induced pluripotent stem cell; LRAT, lecithin retinol acyltransferase; MERTK , C-mer proto-oncogene tyrosine kinase; PAX6 , paired box 6; RDH, retinal dehydrogenase 5; RLBP1, retinaldehyde binding protein 1; RPE, retinal pigment epithelium; TYR, tyrosinase; ZO1, zona occludens protein 1.
Mentions: We used a spontaneous differentiation protocol to generate RPE from wild-type M4C7 and CHM1 iPSCs. Approximately 15 days after the iPSCs were cultured to confluence and the basic fibroblast growth factors were removed from the media, pigmented foci appeared in the plates (Figure 4a). These foci were mechanically passaged, and at confluence, from passage (P) 2, gave rise to a homogeneous layer of polygonal pigmented cells (Figure 4b) characteristic of RPE. Seeding the cells on translucent porous filters allowed sectioning and histological analysis. Observation of semi-thin sections demonstrated that the cell layer was a regular monolayer (Figure 4c). Transmission electron microscopy showed that the monolayer was a polarized epithelium with microvilli on the apical side, a nucleus on the basal side, and cytosolic melanosomes and desmosomes indicative of tight junctions (Figure 4d). The epithelium appeared to secrete a basal lamina detectable between the RPE cells and the filter. Reverse transcriptase–PCR (RT-PCR) studies (Figure 4e) demonstrated that the iPSC-derived epithelium expressed classic genes for the visual cycle (such as RLBP1, RPE65, LRAT, and RDH5), retinal development (PAX6), phagocytosis (MERTK), pigmentation (TYR), ion transport (BEST1), and cell adhesion (ZO-1). Furthermore, IF studies showed that MERTK was localized in the apical microvilli (Figure 4f), CRALBP (Figure 4g) and RPE65 (Figure 4h) in the cytoplasm, and ZO-1 at the apical junctions (Figure 4i), in accordance with their respective roles. Moreover, the presence of desmosomes was consistent with the positive ZO-1-labeling.

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