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• W2102300709 abstract "Research Article23 May 2014Open Access Targeted gene therapy and cell reprogramming in Fanconi anemia Paula Rio Paula Rio Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Rocio Baños Rocio Baños Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Angelo Lombardo Angelo Lombardo San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Oscar Quintana-Bustamante Oscar Quintana-Bustamante Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Lara Alvarez Lara Alvarez Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Zita Garate Zita Garate Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Pietro Genovese Pietro Genovese San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Elena Almarza Elena Almarza Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Antonio Valeri Antonio Valeri Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Begoña Díez Begoña Díez Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Susana Navarro Susana Navarro Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Yaima Torres Yaima Torres NIMGenetics SL, Madrid, Spain Search for more papers by this author Juan P Trujillo Juan P Trujillo Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Universidad Autónoma Barcelona/CIBERER, Barcelona, Spain Search for more papers by this author Rodolfo Murillas Rodolfo Murillas Division of Epithelial Biomedicine, CIEMAT/CIBERER, Madrid, Spain Search for more papers by this author Jose C Segovia Jose C Segovia Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Enrique Samper Enrique Samper NIMGenetics SL, Madrid, Spain Search for more papers by this author Jordi Surralles Jordi Surralles Universidad Autónoma Barcelona/CIBERER, Barcelona, Spain Search for more papers by this author Philip D Gregory Philip D Gregory Sangamo BioSciences Inc., Richmond, CA, USA Search for more papers by this author Michael C Holmes Michael C Holmes Sangamo BioSciences Inc., Richmond, CA, USA Search for more papers by this author Luigi Naldini Corresponding Author Luigi Naldini San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy Vita Salute San Raffaele University, Milan, Italy Search for more papers by this author Juan A Bueren Corresponding Author Juan A Bueren Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Paula Rio Paula Rio Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Rocio Baños Rocio Baños Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Angelo Lombardo Angelo Lombardo San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Oscar Quintana-Bustamante Oscar Quintana-Bustamante Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Lara Alvarez Lara Alvarez Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Zita Garate Zita Garate Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Pietro Genovese Pietro Genovese San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Elena Almarza Elena Almarza Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Antonio Valeri Antonio Valeri Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Begoña Díez Begoña Díez Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Susana Navarro Susana Navarro Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Yaima Torres Yaima Torres NIMGenetics SL, Madrid, Spain Search for more papers by this author Juan P Trujillo Juan P Trujillo Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Universidad Autónoma Barcelona/CIBERER, Barcelona, Spain Search for more papers by this author Rodolfo Murillas Rodolfo Murillas Division of Epithelial Biomedicine, CIEMAT/CIBERER, Madrid, Spain Search for more papers by this author Jose C Segovia Jose C Segovia Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Enrique Samper Enrique Samper NIMGenetics SL, Madrid, Spain Search for more papers by this author Jordi Surralles Jordi Surralles Universidad Autónoma Barcelona/CIBERER, Barcelona, Spain Search for more papers by this author Philip D Gregory Philip D Gregory Sangamo BioSciences Inc., Richmond, CA, USA Search for more papers by this author Michael C Holmes Michael C Holmes Sangamo BioSciences Inc., Richmond, CA, USA Search for more papers by this author Luigi Naldini Corresponding Author Luigi Naldini San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy Vita Salute San Raffaele University, Milan, Italy Search for more papers by this author Juan A Bueren Corresponding Author Juan A Bueren Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Search for more papers by this author Author Information Paula Rio1,2,‡, Rocio Baños1,2,‡, Angelo Lombardo3,‡, Oscar Quintana-Bustamante1,2, Lara Alvarez1,2, Zita Garate1,2, Pietro Genovese3, Elena Almarza1,2, Antonio Valeri1,2, Begoña Díez1,2, Susana Navarro1,2, Yaima Torres4, Juan P Trujillo2,5, Rodolfo Murillas6, Jose C Segovia1,2, Enrique Samper4, Jordi Surralles5, Philip D Gregory7, Michael C Holmes7, Luigi Naldini 3,8 and Juan A Bueren 1,2 1Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain 2Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain 3San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy 4NIMGenetics SL, Madrid, Spain 5Universidad Autónoma Barcelona/CIBERER, Barcelona, Spain 6Division of Epithelial Biomedicine, CIEMAT/CIBERER, Madrid, Spain 7Sangamo BioSciences Inc., Richmond, CA, USA 8Vita Salute San Raffaele University, Milan, Italy ‡These authors contributed equally to this work. *Corresponding author. Tel: +34 913 466 518; Fax: +34 913 466 484; E-mail: juan.bueren@ciemat.es *Corresponding author. Tel: +02 2643 4681; Fax: +02 2643 4621; E-mail: naldini.luigi@hsr.it EMBO Mol Med (2014)6:835-848https://doi.org/10.15252/emmm.201303374 AbstractSynopsis Introduction Results Discussion Materials and Methods Acknowledgements Author contributions Conflict of interest The paper explained For more informationSupporting InformationReferencesPDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. Metrics24MetricsTotal downloads1,492Last 6 Months234Total citations24Last 6 Months1View all metrics ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies. Synopsis This study shows for the first time the possibility of performing targeted gene therapy in a DNA-repair deficiency syndrome, known as Fanconi anemia. By reprogramming targeted cells, asymptomatic gene-edited iPSCs and hematopoietic progenitor cells are generated. Fanconi anemia cells of the FA-A subtype have been efficiently targeted with zinc finger nucleases and a donor construct harboring the therapeutic FANCA gene. The specific insertion of FANCA in the AAVS1 “safe harbor locus” of FA-A fibroblasts efficiently corrected the genetic instability characteristic of these cells. The reprogramming and re-differentiation of gene-edited FA-A fibroblasts generated disease-free hematopoietic progenitors. Introduction The progressive development of engineered nucleases has markedly improved the efficacy and specificity of targeted gene therapy, opening new possibilities for the treatment of inherited and acquired diseases in the clinics (Tebas et al, 2014). In contrast to conventional gene therapy with integrative vectors, targeted gene therapy enables the insertion of foreign sequences (i.e., therapeutic genes or small oligonucleotides) in specific sites of the cell genome. Thus, depending on the genetic etiology of the disease, the gene-targeting approach may pursue the correction of a specific mutation or, alternatively, the insertion of the therapeutic transgene into safe loci of the genome, often referred to as ‘safe harbors’ (Naldini, 2011). In spite of the advances in the field, the question of whether or not targeted gene therapy will be applicable to diseases where homology-directed repair (HDR) is affected has never been explored. Taking into account that Fanconi anemia (FA) proteins participate in HDR (Taniguchi et al, 2002; Yamamoto et al, 2003; Niedzwiedz et al, 2004; Yang et al, 2005; Nakanishi et al, 2011) and coordinate the action of multiple DNA repair processes, including the action of different nucleases and homologous recombination (see reviews in Kee & D'Andrea, 2010; Kottemann & Smogorzewska, 2013; Moldovan & D'Andrea, 2009), we aimed to investigate for the first time the possibility of conducting a targeted gene therapy strategy in FA cells. Genetically, FA is a complex disease where mutations in sixteen different genes (FANC-A, -B, -C, -D1/BRCA2, -D2, -E, -F, -G, -I, -J/BRIP1, -L –M, –N/PALB2, -O/RAD51C; -P/SLX4; -Q/ERCC4/XPF) have been reported (Bogliolo et al, 2013). Among all these genes, mutations in FANCA account for about 60% of total FA patients (Casado et al, 2007; Auerbach, 2009). Importantly, while few recurrent mutations (i.e., truncation of exon 4 in Spanish gypsies or mutations in exons 13, 36, and 38) have been observed in FA-A patients, FANCA mutations are generally private mutations, which include point mutations, microinsertions, microdeletions, splicing mutations and large intragenic deletions (Castella et al, 2011). Thus, considering the large number of genes and mutations that can account for the FA disease, the insertion of a functional FA gene in a ‘safe harbor’ locus would lead to the generation of a targeted gene addition platform with a broad application in FA, regardless of the complementation group and mutation type of each patient. Recent studies by our group and others aiming at the identification of ‘safe harbor sites’ in the human genome have shown robust and stable expression of transgenes integrated in the human PPP1R12C gene, a locus also known as AAVS1, across different cell types (Smith et al, 2008; Lombardo et al, 2011). Additionally, no detectable transcriptional perturbations of the PPP1R12C and its flanking genes were observed after integration of transgenes in this locus, indicating that AAVS1 may represent a safe landing path for therapeutic transgene insertion in the human genome (Lombardo et al, 2011). These observations, together with the development of artificial zinc finger nucleases (ZFNs) that efficiently and selectively target the AAVS1 locus, have facilitated gene editing strategies aiming at inserting therapeutic transgenes in this locus, not only in immortalized cell lines but also in several primary human cell types, including induced pluripotent stem cells (hiPSCs; Hockemeyer et al, 2009; DeKelver et al, 2010; Lombardo et al, 2011; Zou et al, 2011b; Chang & Bouhassira, 2012). Because a defective FA pathway not only predisposes FA patients to cancer (Rosenberg et al, 2008) but also to the early development of bone marrow failure due to the progressive extinction of the HSCs (Larghero et al, 2002; Jacome et al, 2006), our final aim in these studies was the generation of gene-edited, disease-free FA-HSCs, obtained from non-hematopoietic tissues of the patient. Thus, in our current studies, we firstly pursued the specific insertion of the therapeutic FANCA gene in the AAVS1 locus of FA-A patients' fibroblasts. Thereafter, gene-edited FA cells were reprogrammed to generate self-renewing disease-free iPSCs and finally re-differentiated toward the hematopoietic lineage, as previously described with FA cells corrected by conventional LV-mediated gene therapy (Raya et al, 2009). Our goal of conducting a combined approach of gene editing and cell reprogramming in FA cells was particularly challenging taking into account the relevance of the FA pathway both in HDR (Taniguchi et al, 2002; Yamamoto et al, 2003; Niedzwiedz et al, 2004; Yang et al, 2005; Moldovan & D'Andrea, 2009; Kee & D'Andrea, 2010; Nakanishi et al, 2011; Kottemann & Smogorzewska, 2013) and cell reprogramming (Raya et al, 2009; Muller et al, 2012; Yung et al, 2013). In spite of these hurdles, the strong selective growth advantage characteristic of corrected FA cells allowed us to establish a new approach for the efficient generation of FA HPCs harboring specific integrations of the therapeutic FANCA gene in a safe harbor locus. Results Efficient gene-targeting-mediated complementation of fibroblasts from FA-A patients To promote insertion of a FANCA expression cassette into the AAVS1 locus, an integrase-defective lentiviral vector (IDLV) harboring the EGFP and FANCA transgenes flanked by AAVS1 homology arms (donor IDLV) was generated (Fig 1A top). In this donor IDLV, FANCA is under the transcriptional control of the human PGK promoter. In addition, a promoterless EGFP cDNA preceded by a splice acceptor (SA) site and a translational self-cleaving 2A sequence was also included upstream of the FANCA cassette. Upon targeted-mediated insertion into AAVS1, the EGFP cassette will be placed under the transcriptional control of the promoter of the ubiquitously expressed PPP1R12C gene, thus allowing the FACSorting of gene-targeted cells (Fig 1A). Besides the donor IDLV, an adenoviral vector expressing a ZFN pair (AdV5/35-ZFN), designed to induce a DNA double-strand break in the AAVS1 locus, was used to enhance the efficiency of gene targeting in this locus (Hockemeyer et al, 2009). Figure 1. Efficacy of gene targeting of FANCA in the AAVS1 locus of primary hFA-A fibroblasts Top: schematic representation of the donor integrase-defective lentiviral vector (IDLV) used to promote insertion of the EGFP/FANCA cassette into the AAVS1 locus. Middle: AAVS1 locus with the zinc finger nucleases (ZFNs) target site. Bottom: AAVS1 locus upon ZFN-mediated targeted insertion of the EGFP/PGK-FANCA cassette. Black arrow shows transcription of the EGFP from the endogenous PPP1R12C promoter. HA, homology arm; SD, splice donor; SA, splice acceptor; BGHpA, bovine growth hormone polyadenylation signal; SV40pA, simian virus 40 polyadenylation signal. Constituents of the LTR (U5-R-ΔU3) are also indicated. Proliferation advantage of targeted Fanconi anemia (FA) fibroblasts (EGFP+ cells) during in vitro incubation. Comparative analysis of gene targeting in FA-A fibroblasts, untransduced or transduced with a lentiviral vector expressing hTERT. Analyses were performed 14 days after gene targeting. In vitro proliferation advantage of targeted FA fibroblasts (EGFP+) previously transduced with hTERT (FA-52T fibroblasts). Targeted integration analysis of the EGFP/PGK-FANCA cassette into the AAVS1 site by PCR using primers specific for the 5′ or 3′ integration junctions (red arrows in the top schematic) defined as 5′ TI or 3′ TI, respectively. Download figure Download PowerPoint To investigate the feasibility of performing gene targeting in FA-A cells, skin fibroblasts from four FA-A patients with different mutations in FANCA were transduced either with the donor IDLV alone, or with the donor IDLV and the AdV5/35-ZFNs simultaneously. Fourteen days after transduction, cells were analyzed by flow cytometry to measure the proportion of EGFP+ fibroblasts. While <0.05% of the cells transduced with the donor IDLV alone were positive for EGFP, 0.2–1.1% of FA fibroblasts that had been co-transduced with the donor IDLV and the ZFNs-AdV were EGFP+ (See Fig 1B and representative analyses in Supplementary Fig S1). Strikingly, the percentage of EGFP+ cells markedly increased during the in vitro culture of these cells, reaching levels between 5.5 and 13.4% (Fig 1B), showing the proliferation advantage of gene-edited FA-A fibroblasts. Because the prolonged in vitro culture of FA fibroblasts results in increased rates of cell senescence (Muller et al, 2012), in a new set of experiments, fibroblasts from three FA patients (FA-52, FA-123 and FA-644) were transduced with an excisable hTERT-expressing LV (Salmon et al, 2000) prior to performing the gene-targeting procedure. Transduction of FA fibroblasts with hTERT-LVs resulted in a marked increase in telomerase activity (see representative data in Supplementary Fig S2). Significantly, the proportion of EGFP+ cells was markedly increased (3–4-fold) in hTERT-transduced versus untransduced FA fibroblasts from FA patients (Fig 1C), indicating that hTERT improved the efficacy of gene targeting in FA-A fibroblasts. Consistent with data obtained with non-immortalized fibroblasts, when immortalized gene-edited FA fibroblasts were maintained in culture, a progressive increase in the proportion of EGFP+ cells was also observed (see data from geFA-52T in Fig 1D). Strikingly, around 40% of treated FA-A fibroblasts were EGFP+ after 42 days in culture in the absence of any selectable drug (Fig 1D). PCR analyses with two pairs of primers that amplify, respectively, the 5′ and the 3′ integration junctions between the EGFP/FANCA cassette and the endogenous AAVS1 locus evidenced the insertion of the EGFP/FANCA cassette into the AAVS1 locus of sorted EGFP+ geFA-52T fibroblasts (Fig 1E). In these gene-edited FA fibroblasts, the activity of hTERT was also confirmed (Supplementary Fig S2). To investigate whether the insertion of the therapeutic hFANCA cassette in the AAVS1 locus of FA-A fibroblasts corrected the cellular phenotype of the disease, the functionality of the FA pathway in FA-52T fibroblasts was tested both before (negative control) and after the gene-targeting procedure. As a positive control, healthy donor fibroblasts (H.D. Fib) were analyzed in parallel. The presence of nuclear FANCD2 foci, fully dependent on the expression of all the FA core complex proteins, including FANCA (Garcia-Higuera et al, 2001), was determined in these samples after DNA damage induced by mitomycin C (MMC). In contrast to uncorrected FA-52T fibroblasts (FA-52T Fib.), which did not generate FANCD2 foci even after MMC exposure, a significant proportion of the geFA-52T fibroblasts generated FANCD2 foci, mainly after treatment with MMC, thus mimicking the response of H.D. fibroblasts (Fig 2A). Because the main characteristic of FA cells is the increased chromosomal instability upon exposure to DNA inter-strand cross-linking (ICL) drugs, we also investigated the response of both uncorrected and gene-edited FA-A fibroblasts to diepoxybutane (DEB). While in FA-52T fibroblasts DEB induced a significant increase in the number of chromosomal aberrations per cell (from 0.05 ± 0.05 to 1.7 ± 0.46 aberrations/cell)— including chromatid breaks and radial chromosomes, typically found in FA patients′ cells—the same DEB treatment did not induce any increase in the number of chromosomal aberrations in geFA-52T fibroblasts (Fig 2B). Figure 2. Phenotypic correction of the gene-edited FA-A fibroblasts Top: histogram showing the percentage of FA-A fibroblasts, unstransduced or co-transduced with the donor integrase-defective lentiviral vector (IDLV) and the AdV5/35-ZFNs (geFA-52T Fib), showing FANCD2 foci in the absence or the presence of mitomycin C (MMC). Bottom: representative images of FANCD2 foci (red) in cells shown in the top histogram, after MMC treatment. Chromosomal instability induced by diepoxybutane (DEB) in untreated (FA-52T) and gene-edited FA fibroblasts (geFA-52T Fib). Left: representative FISH analysis was performed by staining telomeres (in green), centromeres (in pink) and chromosomes (in blue). Right: histogram showing the number of chromosomal aberrations per cell. Data information: Values are shown as mean ± s.e. from three independent experiments (A) or analysis of twenty different metaphases per group (B). All P-values were calculated using two-tailed unpaired Student's t-test. Download figure Download PowerPoint Taken together, these results show the feasibility of correcting the phenotype of FA cells using gene targeting strategies, in particular by promoting the insertion and expression of FANCA in the AAVS1 safe harbor locus of fibroblasts from FA-A patients. Efficient generation of disease-free iPSCs from FA fibroblasts corrected by gene targeting To generate disease-free FA-iPSCs, FA fibroblasts subjected to gene editing (geFA-123, geFA-52 and geFA-52T) were first enriched for EGFP+ cells by cell sorting and then reprogrammed using a polycistronic excisable LV expressing the human SOX2, OCT4, KLF4, and cMYC transgenes from the EF1A promoter (STEMCCA vector; Somers et al, 2010). Consistent with previous observations (Raya et al, 2009), uncorrected FA fibroblasts did not generate iPSCs after reprogramming, even after transduction with the TERT-LV (data not shown). Although several iPSC-like colonies were generated from gene-edited FA-123 fibroblasts (115 AP+ cells/100,000 fibroblasts), no stable iPSC lines could be generated from FA fibroblasts simply subjected to gene editing, most probably because of the pro-senescence nature of these cells. In marked contrast to these observations, the reprogramming of FA fibroblasts that were first transduced with the hTERT-LV and then subjected to gene editing generated 230 iPSC-like clones, most of which could be maintained after serial in vitro passages (Supplementary Fig S3). Twelve iPSC clones generated from geFA-52T fibroblasts were further expanded and differentiated into fibroblasts to perform additional studies to confirm the integration site of the EGFP/FANCA construct. First, qPCR analyses were conducted to determine the mean copy number per cell of the EGFP/FANCA cassette. As shown in Supplementary Table S1, 11 out of the 12 geFA-iPSC clones analyzed were positive for EGFP integration and contained an average of 0.98 ± 0.44 EGFP copies per cell. The only iPSC clone that did not harbor any EGFP copy (clone 5) did not progress more than six passages in culture. To investigate whether the EGFP/FANCA cassette was specifically integrated in the AAVS1 locus of all these iPSC clones, 3′ primers previously used in analyses of Fig 1E were used. As shown in Supplementary Table S1, all iPSC clones that were positive for integration of the cassette were also positive for the PCR band corresponding to the specific insertion in the AAVS1 locus. Three geFA-iPSC clones (clones 16, 26 and 31) were selected for further characterization. The pluripotency of these gene-corrected clones was first analyzed both by alkaline phosphatase (AP) staining and immunohistochemistry staining of different pluripotency genes. Representative pictures in Fig 3A and Supplementary Fig S4A showed that all tested geFA-iPSCs clones were highly positive for AP, NANOG, TRA-1-60, OCT4, and SSEA-4 expression. RT-qPCR analyses of the expression of endogenous pluripotency genes NANOG, OCT4, SOX2, KLF4, and cMYC were consistent with the pluripotent nature of these clones (Supplementary Fig S4B). In all cases, a very low expression of the ectopic reprogramming transgenes was found, indicating substantial inactivation of the EF1A promoter present in the reprogramming vector. As expected for bona fide iPSC clones, OCT4 and NANOG promoters were hypomethylated in gene-corrected FA-iPSC clones, in clear contrast to the high level of methylation observed in H.D. fibroblasts (Supplementary Fig S4C). To further demonstrate the pluripotency of geFA-iPSC16 cells in vivo, cells were subcutaneously inoculated in NSG mice. Characteristic teratomas containing complex structures representing the three embryonic germ layers were observed 8–10 weeks after implantation. Immunofluorescence staining confirmed the expression of definitive endoderm markers (Fox2A), neural structures that expressed neuroectodermal markers (ß-III-tubulin) and the generation of mesoderm (Brachyury) and mesoderm derivatives tissue such as muscle (α-SMA; Fig 3B). Figure 3. Pluripotency characterization and insertion site analyses of gene-edited FA-A iPSCs Expression of TRA1-60, SSEA-4, OCT4, and NANOG pluripotency markers by immunofluorescence staining of gene-edited FA-iPSCs (geFA-iPSCs; clone 16). Immunofluorescence analysis of ectoderm (β-II-tubulin), endoderm (Fox2A), and mesoderm (α-SMA and Brachyury) in teratomas generated from geFA-iPSCs (clone 16). Southern blot analysis of genomic DNA extracted from the indicated gene-corrected FA iPSC clones (geFA-IPSCs) and from parental fibroblasts, either unmanipulated (FA) or after gene editing (ge-FA iPSCs, clones 16, 26 and 31). Genomic DNA was digested with BglI and hybridized with a probe for PPP1R12C. The band of 9.6 kb corresponds to the targeted integration in PPP1R12C, while the 3.3 kb correspond to the untargeted allele. Southern blot analysis of samples shown in (C) digested with BstXI and hybridized with a probe (P) for EGFP. One single band of 5.1 kb is expected for specific integrations in PPP1R12C. Download figure Download PowerPoint To confirm the insertion of the FANCA cassette into the AAVS1 locus in the gene-corrected FA-iPSC clones, Southern blot analyses were performed on" @default.
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• W2102300709 title "Targeted gene therapy and cell reprogramming in <scp>F</scp> anconi anemia" @default.
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