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

Restoration of a normal cellular phenotype in CHM1 RPE following transduction with AAV2/5-CAG-CHM. (a) A representative in vitro prenylation, followed by western blot analysis of incorporated biotinylated prenyl donor in wild-type (WT), nontransduced (NT) CHM1 RPE, and CHM1 RPE transduced with 100,000 vector genomes (vg) per cell of AAV2/5-CAG-CHM. (b) Normalization to β-actin loading levels and semiquantification indicate that the biotinylated Rab content in wild-type RPE is significantly lower (*P < 0.05) than that of nontransduced CHM1 RPE (data expressed as mean ± SEM, n = 3). Following transduction of CHM1 RPE with AAV2/5-CAG-CHM, the biotinylated Rab pool was significantly reduced (*P < 0.05) to levels not significantly different from wild-type levels. (c) Differential centrifugation and western blot analysis of cytosolic and membrane fractions in wild-type (WT), nontransduced (NT) CHM1 RPE, and CHM1 RPE transduced with 100,000 vg per cell of AAV2/5-CAG-CHM or AAV2/5-CAG-EGFP. (d) Semiquantification analysis indicates that wild-type cytosolic Rab27A levels were significantly different (*P < 0.05) from the Rab27A levels in nontransduced CHM1. Following transduction of CHM1 RPE with AAV2/5-CAG-CHM, the cytosolic Rab27A content was significantly reduced (*P < 0.05) to levels not significantly different from the wild-type levels (data expressed as mean ± SEM, n = 3). By contrast, Rab27A cytosolic levels in CHM1 RPE were unchanged following transduction with AAV2/5-CAG-EGFP. AAV, adeno-associated virus; CAG, chicken β-actin with a CMV enhancer; RPE, retinal pigment epithelium.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig8: Restoration of a normal cellular phenotype in CHM1 RPE following transduction with AAV2/5-CAG-CHM. (a) A representative in vitro prenylation, followed by western blot analysis of incorporated biotinylated prenyl donor in wild-type (WT), nontransduced (NT) CHM1 RPE, and CHM1 RPE transduced with 100,000 vector genomes (vg) per cell of AAV2/5-CAG-CHM. (b) Normalization to β-actin loading levels and semiquantification indicate that the biotinylated Rab content in wild-type RPE is significantly lower (*P < 0.05) than that of nontransduced CHM1 RPE (data expressed as mean ± SEM, n = 3). Following transduction of CHM1 RPE with AAV2/5-CAG-CHM, the biotinylated Rab pool was significantly reduced (*P < 0.05) to levels not significantly different from wild-type levels. (c) Differential centrifugation and western blot analysis of cytosolic and membrane fractions in wild-type (WT), nontransduced (NT) CHM1 RPE, and CHM1 RPE transduced with 100,000 vg per cell of AAV2/5-CAG-CHM or AAV2/5-CAG-EGFP. (d) Semiquantification analysis indicates that wild-type cytosolic Rab27A levels were significantly different (*P < 0.05) from the Rab27A levels in nontransduced CHM1. Following transduction of CHM1 RPE with AAV2/5-CAG-CHM, the cytosolic Rab27A content was significantly reduced (*P < 0.05) to levels not significantly different from the wild-type levels (data expressed as mean ± SEM, n = 3). By contrast, Rab27A cytosolic levels in CHM1 RPE were unchanged following transduction with AAV2/5-CAG-EGFP. AAV, adeno-associated virus; CAG, chicken β-actin with a CMV enhancer; RPE, retinal pigment epithelium.

Mentions: CHM1 RPE was transduced with AAV2/5-CAG-CHM vector and, based on the results of the time-course experiments, REP1 activity was analyzed 4 weeks posttransduction. A representative experiment is shown in Figure 8a. Following transduction of the CHM1 RPE with the AAV2/5-CAG-CHM vector and semiquantification of three experiments (Figure 8b), we detected a significant reduction in the quantity of biotinylated Rab proteins (P < 0.05) to levels that were not significantly different from those of wild-type RPE. Similarly, an analysis of the subcellular distribution of Rab27A following AAV2/5-CAG-CHM transduction was performed, and a representative experiment is shown in Figure 8c. The semiquantification of three experiments (Figure 8d) showed that the cytosolic versus membrane-bound Rab27A content in transduced CHM1 RPE was significantly reduced (P < 0.05) to levels similar to wild-type RPE. By contrast, transduction with a control AAV2/5-CAG-EGFP vector did not alter the proportions of Rab27A as compared with nontransduced CHM1 RPE (Figure 8d). A parallel transduction with AAV2/5-CAG-EGFP, analyzed by flow cytometry, suggested a transduction efficiency of ~70% in these experiments.


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)

Restoration of a normal cellular phenotype in CHM1 RPE following transduction with AAV2/5-CAG-CHM. (a) A representative in vitro prenylation, followed by western blot analysis of incorporated biotinylated prenyl donor in wild-type (WT), nontransduced (NT) CHM1 RPE, and CHM1 RPE transduced with 100,000 vector genomes (vg) per cell of AAV2/5-CAG-CHM. (b) Normalization to β-actin loading levels and semiquantification indicate that the biotinylated Rab content in wild-type RPE is significantly lower (*P < 0.05) than that of nontransduced CHM1 RPE (data expressed as mean ± SEM, n = 3). Following transduction of CHM1 RPE with AAV2/5-CAG-CHM, the biotinylated Rab pool was significantly reduced (*P < 0.05) to levels not significantly different from wild-type levels. (c) Differential centrifugation and western blot analysis of cytosolic and membrane fractions in wild-type (WT), nontransduced (NT) CHM1 RPE, and CHM1 RPE transduced with 100,000 vg per cell of AAV2/5-CAG-CHM or AAV2/5-CAG-EGFP. (d) Semiquantification analysis indicates that wild-type cytosolic Rab27A levels were significantly different (*P < 0.05) from the Rab27A levels in nontransduced CHM1. Following transduction of CHM1 RPE with AAV2/5-CAG-CHM, the cytosolic Rab27A content was significantly reduced (*P < 0.05) to levels not significantly different from the wild-type levels (data expressed as mean ± SEM, n = 3). By contrast, Rab27A cytosolic levels in CHM1 RPE were unchanged following transduction with AAV2/5-CAG-EGFP. AAV, adeno-associated virus; CAG, chicken β-actin with a CMV enhancer; RPE, retinal pigment epithelium.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig8: Restoration of a normal cellular phenotype in CHM1 RPE following transduction with AAV2/5-CAG-CHM. (a) A representative in vitro prenylation, followed by western blot analysis of incorporated biotinylated prenyl donor in wild-type (WT), nontransduced (NT) CHM1 RPE, and CHM1 RPE transduced with 100,000 vector genomes (vg) per cell of AAV2/5-CAG-CHM. (b) Normalization to β-actin loading levels and semiquantification indicate that the biotinylated Rab content in wild-type RPE is significantly lower (*P < 0.05) than that of nontransduced CHM1 RPE (data expressed as mean ± SEM, n = 3). Following transduction of CHM1 RPE with AAV2/5-CAG-CHM, the biotinylated Rab pool was significantly reduced (*P < 0.05) to levels not significantly different from wild-type levels. (c) Differential centrifugation and western blot analysis of cytosolic and membrane fractions in wild-type (WT), nontransduced (NT) CHM1 RPE, and CHM1 RPE transduced with 100,000 vg per cell of AAV2/5-CAG-CHM or AAV2/5-CAG-EGFP. (d) Semiquantification analysis indicates that wild-type cytosolic Rab27A levels were significantly different (*P < 0.05) from the Rab27A levels in nontransduced CHM1. Following transduction of CHM1 RPE with AAV2/5-CAG-CHM, the cytosolic Rab27A content was significantly reduced (*P < 0.05) to levels not significantly different from the wild-type levels (data expressed as mean ± SEM, n = 3). By contrast, Rab27A cytosolic levels in CHM1 RPE were unchanged following transduction with AAV2/5-CAG-EGFP. AAV, adeno-associated virus; CAG, chicken β-actin with a CMV enhancer; RPE, retinal pigment epithelium.
Mentions: CHM1 RPE was transduced with AAV2/5-CAG-CHM vector and, based on the results of the time-course experiments, REP1 activity was analyzed 4 weeks posttransduction. A representative experiment is shown in Figure 8a. Following transduction of the CHM1 RPE with the AAV2/5-CAG-CHM vector and semiquantification of three experiments (Figure 8b), we detected a significant reduction in the quantity of biotinylated Rab proteins (P < 0.05) to levels that were not significantly different from those of wild-type RPE. Similarly, an analysis of the subcellular distribution of Rab27A following AAV2/5-CAG-CHM transduction was performed, and a representative experiment is shown in Figure 8c. The semiquantification of three experiments (Figure 8d) showed that the cytosolic versus membrane-bound Rab27A content in transduced CHM1 RPE was significantly reduced (P < 0.05) to levels similar to wild-type RPE. By contrast, transduction with a control AAV2/5-CAG-EGFP vector did not alter the proportions of Rab27A as compared with nontransduced CHM1 RPE (Figure 8d). A parallel transduction with AAV2/5-CAG-EGFP, analyzed by flow cytometry, suggested a transduction efficiency of ~70% in these experiments.

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