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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.


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Generation of CHM1 iPSCs. (a) Fibroblasts of patient CHM1 in culture. Bar = 100 µm. (b) Lentiviral transduction and doxycycline induction results in the expression of the four reprogramming factors, as determined by quantitative polymerase chain reaction analysis. (c) Successful reprogramming results in the detection of an iPSC colony composed of tightly packed cells with a clear border separating them from the underlying fibroblasts. Bar = 200 µm. (d) The deletion in CHM1 results in the amplification of exons 7 and 9 in patient fibroblasts (Fib) and iPSCs, but in the absence of exon 8, as opposed to the situation in wild-type (WT) DNA; − indicates negative control. (e) At the complementary DNA (cDNA) level, the CHM1 transcript is 226 bp smaller, corresponding to the deletion of exon 8. (f) No large chromosomal anomalies in CHM1 iPSCs were detected by karyotype analysis. iPSC, induced pluripotent stem cell.
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fig2: Generation of CHM1 iPSCs. (a) Fibroblasts of patient CHM1 in culture. Bar = 100 µm. (b) Lentiviral transduction and doxycycline induction results in the expression of the four reprogramming factors, as determined by quantitative polymerase chain reaction analysis. (c) Successful reprogramming results in the detection of an iPSC colony composed of tightly packed cells with a clear border separating them from the underlying fibroblasts. Bar = 200 µm. (d) The deletion in CHM1 results in the amplification of exons 7 and 9 in patient fibroblasts (Fib) and iPSCs, but in the absence of exon 8, as opposed to the situation in wild-type (WT) DNA; − indicates negative control. (e) At the complementary DNA (cDNA) level, the CHM1 transcript is 226 bp smaller, corresponding to the deletion of exon 8. (f) No large chromosomal anomalies in CHM1 iPSCs were detected by karyotype analysis. iPSC, induced pluripotent stem cell.

Mentions: We used doxycycline-inducible lentiviral vectors carrying the Yamanaka transcription factor cocktail (c-MYC, KLF4, OCT4, and SOX2) to reprogram CHM1 fibroblasts (Figure 2a). We verified the expression of each transgene 24 hours after doxycycline induction by quantitative polymerase chain reaction (q-PCR) studies (Figure 2b). One week after doxycycline induction, the fibroblasts began to change morphology, with partially reprogrammed colonies appearing and disappearing over time. By contrast, 5 weeks after doxycycline induction, a morphologically characteristic iPSC colony was detected (Figure 2c), which survived mechanical passaging into embryonic stem (ES) cell media without doxycycline. PCR amplification of CHM1 iPSC DNA showed that the original CHM deletion was present (Figure 2d) and that it led to the complete deletion of exon 8 from the mRNA, as evidenced by a difference of 226 bp between the complementary DNA (cDNA) fragments generated from wild-type and CHM1 iPSC RNAs (Figure 2e). In addition, the CHM1 iPSCs did not present any large chromosomal anomalies that may occur during reprogramming, as shown by karyotype analysis (Figure 2f).


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 of CHM1 iPSCs. (a) Fibroblasts of patient CHM1 in culture. Bar = 100 µm. (b) Lentiviral transduction and doxycycline induction results in the expression of the four reprogramming factors, as determined by quantitative polymerase chain reaction analysis. (c) Successful reprogramming results in the detection of an iPSC colony composed of tightly packed cells with a clear border separating them from the underlying fibroblasts. Bar = 200 µm. (d) The deletion in CHM1 results in the amplification of exons 7 and 9 in patient fibroblasts (Fib) and iPSCs, but in the absence of exon 8, as opposed to the situation in wild-type (WT) DNA; − indicates negative control. (e) At the complementary DNA (cDNA) level, the CHM1 transcript is 226 bp smaller, corresponding to the deletion of exon 8. (f) No large chromosomal anomalies in CHM1 iPSCs were detected by karyotype analysis. iPSC, induced pluripotent stem cell.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Generation of CHM1 iPSCs. (a) Fibroblasts of patient CHM1 in culture. Bar = 100 µm. (b) Lentiviral transduction and doxycycline induction results in the expression of the four reprogramming factors, as determined by quantitative polymerase chain reaction analysis. (c) Successful reprogramming results in the detection of an iPSC colony composed of tightly packed cells with a clear border separating them from the underlying fibroblasts. Bar = 200 µm. (d) The deletion in CHM1 results in the amplification of exons 7 and 9 in patient fibroblasts (Fib) and iPSCs, but in the absence of exon 8, as opposed to the situation in wild-type (WT) DNA; − indicates negative control. (e) At the complementary DNA (cDNA) level, the CHM1 transcript is 226 bp smaller, corresponding to the deletion of exon 8. (f) No large chromosomal anomalies in CHM1 iPSCs were detected by karyotype analysis. iPSC, induced pluripotent stem cell.
Mentions: We used doxycycline-inducible lentiviral vectors carrying the Yamanaka transcription factor cocktail (c-MYC, KLF4, OCT4, and SOX2) to reprogram CHM1 fibroblasts (Figure 2a). We verified the expression of each transgene 24 hours after doxycycline induction by quantitative polymerase chain reaction (q-PCR) studies (Figure 2b). One week after doxycycline induction, the fibroblasts began to change morphology, with partially reprogrammed colonies appearing and disappearing over time. By contrast, 5 weeks after doxycycline induction, a morphologically characteristic iPSC colony was detected (Figure 2c), which survived mechanical passaging into embryonic stem (ES) cell media without doxycycline. PCR amplification of CHM1 iPSC DNA showed that the original CHM deletion was present (Figure 2d) and that it led to the complete deletion of exon 8 from the mRNA, as evidenced by a difference of 226 bp between the complementary DNA (cDNA) fragments generated from wild-type and CHM1 iPSC RNAs (Figure 2e). In addition, the CHM1 iPSCs did not present any large chromosomal anomalies that may occur during reprogramming, as shown by karyotype analysis (Figure 2f).

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