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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

Characterization of CHM1 iPSCs. (a) Downregulation of the expression of the pluripotency genes delivered by the lentiviral vectors in iPSCs, compared with the expression in transduced fibroblasts (Tr Fib), as detected by q-PCR analysis. (b) Activation of the host’s endogenous pluripotency genes in iPSCs, as compared with fibroblasts (Fib), as shown by q-PCR analysis. (c) Expression of the pluripotency marker alkaline phosphatase by cell staining. (d) Magnification of the colony boxed in c. Bar = 500 µm. (e) Expression of the nuclear pluripotency marker NANOG (in green), as shown by immunofluorescence studies and merged with a Hoechst staining of the nuclei (in blue). Bar = 50 µm. Inset, nuclear expression of NANOG exclusively. (f) Expression of the pluripotency marker SSEA3 (in red) merged with Hoechst staining of the nuclei. Bar = 200 µm. Subcutaneous injection of CHM1 iPSCs into immunodeficient mice results in the formation of (g) teratomas that contain derivatives of the three germ lines: (h) neuroblastic rosettes (arrows) that are ectodermal in origin (bar = 50 µm); (i) osteocytes (arrow) that are mesodermal in origin (bar = 100 µm), and (j) adipose cells (arrows) that are endodermal in origin (bar = 100 µm). iPSC, induced pluripotent stem cell; OCT4, octamer binding transcription factor 4; SOX2, sex determining region Y-box 2.
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fig3: Characterization of CHM1 iPSCs. (a) Downregulation of the expression of the pluripotency genes delivered by the lentiviral vectors in iPSCs, compared with the expression in transduced fibroblasts (Tr Fib), as detected by q-PCR analysis. (b) Activation of the host’s endogenous pluripotency genes in iPSCs, as compared with fibroblasts (Fib), as shown by q-PCR analysis. (c) Expression of the pluripotency marker alkaline phosphatase by cell staining. (d) Magnification of the colony boxed in c. Bar = 500 µm. (e) Expression of the nuclear pluripotency marker NANOG (in green), as shown by immunofluorescence studies and merged with a Hoechst staining of the nuclei (in blue). Bar = 50 µm. Inset, nuclear expression of NANOG exclusively. (f) Expression of the pluripotency marker SSEA3 (in red) merged with Hoechst staining of the nuclei. Bar = 200 µm. Subcutaneous injection of CHM1 iPSCs into immunodeficient mice results in the formation of (g) teratomas that contain derivatives of the three germ lines: (h) neuroblastic rosettes (arrows) that are ectodermal in origin (bar = 50 µm); (i) osteocytes (arrow) that are mesodermal in origin (bar = 100 µm), and (j) adipose cells (arrows) that are endodermal in origin (bar = 100 µm). iPSC, induced pluripotent stem cell; OCT4, octamer binding transcription factor 4; SOX2, sex determining region Y-box 2.

Mentions: The pluripotency of the CHM1 iPSCs was verified by a variety of techniques and using a wild-type clone M4C7 (ref. 27) as a positive control. For a cell to be considered as correctly reprogrammed to a pluripotent state, host pluripotency genes need to be activated, in the absence of expression of exogenous pluripotency genes, and their encoded proteins need to be produced. First, at the mRNA level, q-PCR studies demonstrated the silencing of exogenous c-MYC, KLF4, OCT4, and SOX2 delivered by the lentiviral vectors (Figure 3a) and the activation of expression of endogenous OCT4, SOX2, LIN28, and NANOG in the CHM1 iPSCs (Figure 3b). Second, alkaline phosphatase staining was positive (Figure 3c,d), and immunofluorescence (IF) studies confirmed the expression of NANOG (Figure 3e) and indicated the expression of SSEA3 (Figure 3f). Finally, the CHM1 iPSCs induced the formation of teratomas (Figure 3g) when injected subcutaneously into immunodeficient mice and the presence of derivatives of the three germ layers (ectoderm (Figure 3h), mesoderm (Figure 3i), and endoderm (Figure 3j)) was confirmed by histological analysis.


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 CHM1 iPSCs. (a) Downregulation of the expression of the pluripotency genes delivered by the lentiviral vectors in iPSCs, compared with the expression in transduced fibroblasts (Tr Fib), as detected by q-PCR analysis. (b) Activation of the host’s endogenous pluripotency genes in iPSCs, as compared with fibroblasts (Fib), as shown by q-PCR analysis. (c) Expression of the pluripotency marker alkaline phosphatase by cell staining. (d) Magnification of the colony boxed in c. Bar = 500 µm. (e) Expression of the nuclear pluripotency marker NANOG (in green), as shown by immunofluorescence studies and merged with a Hoechst staining of the nuclei (in blue). Bar = 50 µm. Inset, nuclear expression of NANOG exclusively. (f) Expression of the pluripotency marker SSEA3 (in red) merged with Hoechst staining of the nuclei. Bar = 200 µm. Subcutaneous injection of CHM1 iPSCs into immunodeficient mice results in the formation of (g) teratomas that contain derivatives of the three germ lines: (h) neuroblastic rosettes (arrows) that are ectodermal in origin (bar = 50 µm); (i) osteocytes (arrow) that are mesodermal in origin (bar = 100 µm), and (j) adipose cells (arrows) that are endodermal in origin (bar = 100 µm). iPSC, induced pluripotent stem cell; OCT4, octamer binding transcription factor 4; SOX2, sex determining region Y-box 2.
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Related In: Results  -  Collection

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fig3: Characterization of CHM1 iPSCs. (a) Downregulation of the expression of the pluripotency genes delivered by the lentiviral vectors in iPSCs, compared with the expression in transduced fibroblasts (Tr Fib), as detected by q-PCR analysis. (b) Activation of the host’s endogenous pluripotency genes in iPSCs, as compared with fibroblasts (Fib), as shown by q-PCR analysis. (c) Expression of the pluripotency marker alkaline phosphatase by cell staining. (d) Magnification of the colony boxed in c. Bar = 500 µm. (e) Expression of the nuclear pluripotency marker NANOG (in green), as shown by immunofluorescence studies and merged with a Hoechst staining of the nuclei (in blue). Bar = 50 µm. Inset, nuclear expression of NANOG exclusively. (f) Expression of the pluripotency marker SSEA3 (in red) merged with Hoechst staining of the nuclei. Bar = 200 µm. Subcutaneous injection of CHM1 iPSCs into immunodeficient mice results in the formation of (g) teratomas that contain derivatives of the three germ lines: (h) neuroblastic rosettes (arrows) that are ectodermal in origin (bar = 50 µm); (i) osteocytes (arrow) that are mesodermal in origin (bar = 100 µm), and (j) adipose cells (arrows) that are endodermal in origin (bar = 100 µm). iPSC, induced pluripotent stem cell; OCT4, octamer binding transcription factor 4; SOX2, sex determining region Y-box 2.
Mentions: The pluripotency of the CHM1 iPSCs was verified by a variety of techniques and using a wild-type clone M4C7 (ref. 27) as a positive control. For a cell to be considered as correctly reprogrammed to a pluripotent state, host pluripotency genes need to be activated, in the absence of expression of exogenous pluripotency genes, and their encoded proteins need to be produced. First, at the mRNA level, q-PCR studies demonstrated the silencing of exogenous c-MYC, KLF4, OCT4, and SOX2 delivered by the lentiviral vectors (Figure 3a) and the activation of expression of endogenous OCT4, SOX2, LIN28, and NANOG in the CHM1 iPSCs (Figure 3b). Second, alkaline phosphatase staining was positive (Figure 3c,d), and immunofluorescence (IF) studies confirmed the expression of NANOG (Figure 3e) and indicated the expression of SSEA3 (Figure 3f). Finally, the CHM1 iPSCs induced the formation of teratomas (Figure 3g) when injected subcutaneously into immunodeficient mice and the presence of derivatives of the three germ layers (ectoderm (Figure 3h), mesoderm (Figure 3i), and endoderm (Figure 3j)) was confirmed by histological analysis.

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