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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 the CHM deletion of patient CHM1. (a) The sequence of exon 7 of the CHM gene is indicated in green, and the sequence of exon 8 is indicated in purple. The 7-bp sequence (underlined in green) in exon 7, and the 15-bp sequence in exon 8 (underlined in purple), which are duplicated in the DNA of CHM1 are positioned on the wild-type complementary DNA (cDNA) sequence. The limits of the resulting deletion (arrowheads) are also indicated. The duplicated and deleted cDNA sequences of patient CHM1 for the corresponding region are shown. The inserted sequence is in gray. (b) The wild-type REP1 sequence of 653 aa containing two guanosine diphosphate (GDP) dissociation inhibitor (GDI) domains, indicated in red and blue. The truncated REP1 sequence is predicted to be 332 aa long and to contain a truncated second GDI domain (in blue). (c) Western blot analysis with an antibody recognizing both REP1 and REP2 detects two bands that migrate at ~110 (arrowhead) and 120 (arrow) kDa, respectively, in wild-type cells. In the cells of CHM1, only the larger band corresponding to REP2 (arrow) is detected. This was confirmed using an antibody specific to REP1, which detected a single band in wild-type cells (arrowhead) and no band in CHM1 cells. REP1, Rab escort protein 1.
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fig1: Characterization of the CHM deletion of patient CHM1. (a) The sequence of exon 7 of the CHM gene is indicated in green, and the sequence of exon 8 is indicated in purple. The 7-bp sequence (underlined in green) in exon 7, and the 15-bp sequence in exon 8 (underlined in purple), which are duplicated in the DNA of CHM1 are positioned on the wild-type complementary DNA (cDNA) sequence. The limits of the resulting deletion (arrowheads) are also indicated. The duplicated and deleted cDNA sequences of patient CHM1 for the corresponding region are shown. The inserted sequence is in gray. (b) The wild-type REP1 sequence of 653 aa containing two guanosine diphosphate (GDP) dissociation inhibitor (GDI) domains, indicated in red and blue. The truncated REP1 sequence is predicted to be 332 aa long and to contain a truncated second GDI domain (in blue). (c) Western blot analysis with an antibody recognizing both REP1 and REP2 detects two bands that migrate at ~110 (arrowhead) and 120 (arrow) kDa, respectively, in wild-type cells. In the cells of CHM1, only the larger band corresponding to REP2 (arrow) is detected. This was confirmed using an antibody specific to REP1, which detected a single band in wild-type cells (arrowhead) and no band in CHM1 cells. REP1, Rab escort protein 1.

Mentions: The characterization of genomic DNA from the fibroblasts of patient CHM1 revealed a duplication of a 7-bp (TAGTTCT) sequence in intron 7 situated 40 bp upstream of the start of exon 8. In between the duplicated sequence was a 4-bp insertion (GATT) (Figure 1a). This first duplication was followed by a second duplication of a 15-bp sequence within exon 8 (GTCATGCATTCAATT), situated 68 bp after the start. The 97 bp situated between the two duplicated sequences, which comprised the end of intron 7 and the beginning of exon 8, were deleted. The loss of the intron 7 acceptor splice site and the subsequent deletion of exon 8 resulted in a predicted frameshift at amino acid (aa) position 314 and a premature stop codon at position 332 (wild-type REP1 is 653 aa long), truncating the second guanosine diphosphate dissociation inhibitor domain of REP1 (Figure 1b). Western blot analysis with an antibody directed against an N-terminal epitope present in both wild-type and truncated REP1 (73.49 kDa), as well as in REP2 (74.08 kDa), detected two bands for the control cells and one band for the CHM1 cells (corresponding to REP2), suggesting that the truncated protein was unstable (Figure 1c). Consistently, western blot analysis using a REP1-specific antibody did not detect a protein in CHM1 cells as opposed to the results in control cells


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 CHM deletion of patient CHM1. (a) The sequence of exon 7 of the CHM gene is indicated in green, and the sequence of exon 8 is indicated in purple. The 7-bp sequence (underlined in green) in exon 7, and the 15-bp sequence in exon 8 (underlined in purple), which are duplicated in the DNA of CHM1 are positioned on the wild-type complementary DNA (cDNA) sequence. The limits of the resulting deletion (arrowheads) are also indicated. The duplicated and deleted cDNA sequences of patient CHM1 for the corresponding region are shown. The inserted sequence is in gray. (b) The wild-type REP1 sequence of 653 aa containing two guanosine diphosphate (GDP) dissociation inhibitor (GDI) domains, indicated in red and blue. The truncated REP1 sequence is predicted to be 332 aa long and to contain a truncated second GDI domain (in blue). (c) Western blot analysis with an antibody recognizing both REP1 and REP2 detects two bands that migrate at ~110 (arrowhead) and 120 (arrow) kDa, respectively, in wild-type cells. In the cells of CHM1, only the larger band corresponding to REP2 (arrow) is detected. This was confirmed using an antibody specific to REP1, which detected a single band in wild-type cells (arrowhead) and no band in CHM1 cells. REP1, Rab escort protein 1.
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Related In: Results  -  Collection

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fig1: Characterization of the CHM deletion of patient CHM1. (a) The sequence of exon 7 of the CHM gene is indicated in green, and the sequence of exon 8 is indicated in purple. The 7-bp sequence (underlined in green) in exon 7, and the 15-bp sequence in exon 8 (underlined in purple), which are duplicated in the DNA of CHM1 are positioned on the wild-type complementary DNA (cDNA) sequence. The limits of the resulting deletion (arrowheads) are also indicated. The duplicated and deleted cDNA sequences of patient CHM1 for the corresponding region are shown. The inserted sequence is in gray. (b) The wild-type REP1 sequence of 653 aa containing two guanosine diphosphate (GDP) dissociation inhibitor (GDI) domains, indicated in red and blue. The truncated REP1 sequence is predicted to be 332 aa long and to contain a truncated second GDI domain (in blue). (c) Western blot analysis with an antibody recognizing both REP1 and REP2 detects two bands that migrate at ~110 (arrowhead) and 120 (arrow) kDa, respectively, in wild-type cells. In the cells of CHM1, only the larger band corresponding to REP2 (arrow) is detected. This was confirmed using an antibody specific to REP1, which detected a single band in wild-type cells (arrowhead) and no band in CHM1 cells. REP1, Rab escort protein 1.
Mentions: The characterization of genomic DNA from the fibroblasts of patient CHM1 revealed a duplication of a 7-bp (TAGTTCT) sequence in intron 7 situated 40 bp upstream of the start of exon 8. In between the duplicated sequence was a 4-bp insertion (GATT) (Figure 1a). This first duplication was followed by a second duplication of a 15-bp sequence within exon 8 (GTCATGCATTCAATT), situated 68 bp after the start. The 97 bp situated between the two duplicated sequences, which comprised the end of intron 7 and the beginning of exon 8, were deleted. The loss of the intron 7 acceptor splice site and the subsequent deletion of exon 8 resulted in a predicted frameshift at amino acid (aa) position 314 and a premature stop codon at position 332 (wild-type REP1 is 653 aa long), truncating the second guanosine diphosphate dissociation inhibitor domain of REP1 (Figure 1b). Western blot analysis with an antibody directed against an N-terminal epitope present in both wild-type and truncated REP1 (73.49 kDa), as well as in REP2 (74.08 kDa), detected two bands for the control cells and one band for the CHM1 cells (corresponding to REP2), suggesting that the truncated protein was unstable (Figure 1c). Consistently, western blot analysis using a REP1-specific antibody did not detect a protein in CHM1 cells as opposed to the results in control cells

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