Publications by authors named "Paula Río"

40 Publications

Natural gene therapy by reverse mosaicism leads to improved hematology in Fanconi anemia patients.

Am J Hematol 2021 May 13. Epub 2021 May 13.

Genomic Instability and DNA Repair Syndromes Group and Joint Research Unit on Genomic Medicine UAB-Sant Pau Biomedical Research Institute (IIB Sant Pau), Institut de Recerca Hospital de la Santa Creu i Sant Pau-IIB Sant Pau, Barcelona, Spain.

Fanconi anemia (FA) is characterized by chromosome fragility, bone marrow failure (BMF) and predisposition to cancer. As reverse genetic mosaicism has been described as "natural gene therapy" in patients with FA, we sought to evaluate the clinical course of a cohort of FA mosaic patients followed at referral centers in Spain over a 30-year period. This cohort includes patients with a majority of T cells without chromosomal aberrations in the DEB-chromosomal breakage test. Relative to non-mosaic FA patients, we observed a higher proportion of adult patients in the cohort of mosaics, with a later age of hematologic onset and a milder evolution of (BMF). Consequently, the requirement for hematopoietic stem cell transplant (HSCT) was also lower. Additional studies allowed us to identify a sub-cohort of mosaic FA patients in whom the reversion was present in bone marrow (BM) progenitor cells leading to multilineage mosaicism. These multilineage mosaic patients are older, have a lower percentage of aberrant cells, have more stable hematology and none of them developed leukemia or myelodysplastic syndrome when compared to non-mosaics. In conclusion, our data indicate that reverse mosaicism is a good prognostic factor in FA and is associated with more favorable long-term clinical outcomes.
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http://dx.doi.org/10.1002/ajh.26234DOI Listing
May 2021

Generating New FANCA-Deficient HNSCC Cell Lines by Genomic Editing Recapitulates the Cellular Phenotypes of Fanconi Anemia.

Genes (Basel) 2021 Apr 9;12(4). Epub 2021 Apr 9.

Biomedical Research Institute I+12, University Hospital 12 de Octubre, 28041 Madrid, Spain.

Fanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA patients display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore, better therapies as well as new disease models are urgently needed. We have used CRISPR/Cas9 editing tools in order to interrupt the human gene by the generation of insertions/deletions (indels) in exon 4 in two cancer cell lines from sporadic HNSCC having no mutation in FA-genes: CAL27 and CAL33 cells. Our approach allowed efficient editing, subsequent purification of single-cell clones, and Sanger sequencing validation at the edited locus. Clones having frameshift indels in homozygosis did not express FANCA protein and were selected for further analysis. When compared with parental CAL27 and CAL33, -mutant cell clones displayed a FA-phenotype as they (i) are highly sensitive to DNA interstrand crosslink (ICL) agents such as mitomycin C (MMC) or cisplatin, (ii) do not monoubiquitinate FANCD2 upon MMC treatment and therefore (iii) do not form FANCD2 nuclear foci, and (iv) they display increased chromosome fragility and G2 arrest after diepoxybutane (DEB) treatment. These -mutant clones display similar growth rates as their parental cells. Interestingly, mutant cells acquire phenotypes associated with more aggressive disease, such as increased migration in wound healing assays. Therefore, CAL27 and CAL33 cells with mutations are phenocopies of FA-HNSCC cells.
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http://dx.doi.org/10.3390/genes12040548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8069753PMC
April 2021

Next-generation Sequencing in Bone Marrow Failure Syndromes and Isolated Cytopenias: Experience of the Spanish Network on Bone Marrow Failure Syndromes.

Hemasphere 2021 Apr 9;5(4):e539. Epub 2021 Mar 9.

Servicio de Hematología y Oncología Pediátrica, Fundación de Investigación Biomédica, Hospital Infantil Universitario Niño Jesús, Madrid, Spain.

Inherited bone marrow failure syndromes (IBMFSs) are a group of congenital rare diseases characterized by bone marrow failure, congenital anomalies, high genetic heterogeneity, and predisposition to cancer. Appropriate treatment and cancer surveillance ideally depend on the identification of the mutated gene. A next-generation sequencing (NGS) panel of genes could be 1 initial genetic screening test to be carried out in a comprehensive study of IBMFSs, allowing molecular detection in affected patients. We designed 2 NGS panels of IBMFS genes: version 1 included 129 genes and version 2 involved 145 genes. The cohort included a total of 204 patients with suspected IBMFSs without molecular diagnosis. Capture-based targeted sequencing covered > 99% of the target regions of 145 genes, with more than 20 independent reads. No differences were seen between the 2 versions of the panel. The NGS tool allowed a total of 91 patients to be diagnosed, with an overall molecular diagnostic rate of 44%. Among the 167 patients with classified IBMFSs, 81 patients (48%) were diagnosed. Unclassified IBMFSs involved a total of 37 patients, of whom 9 patients (24%) were diagnosed. The preexisting diagnosis of 6 clinically classified patients (6%) was amended, implying a change of therapy for some of them. Our NGS IBMFS gene panel assay is a useful tool in the molecular diagnosis of IBMFSs and a reasonable option as the first tier genetic test in these disorders.
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http://dx.doi.org/10.1097/HS9.0000000000000539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7951136PMC
April 2021

TALEN mediated gene editing in a mouse model of Fanconi anemia.

Sci Rep 2020 04 24;10(1):6997. Epub 2020 Apr 24.

Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, 28040, Madrid, Spain.

The promising ability to genetically modify hematopoietic stem and progenitor cells by precise gene editing remains challenging due to their sensitivity to in vitro manipulations and poor efficiencies of homologous recombination. This study represents the first evidence of implementing a gene editing strategy in a murine safe harbor locus site that phenotypically corrects primary cells from a mouse model of Fanconi anemia A. By means of the co-delivery of transcription activator-like effector nucleases and a donor therapeutic FANCA template to the Mbs85 locus, we achieved efficient gene targeting (23%) in mFA-A fibroblasts. This resulted in the phenotypic correction of these cells, as revealed by the reduced sensitivity of these cells to mitomycin C. Moreover, robust evidence of targeted integration was observed in murine wild type and FA-A hematopoietic progenitor cells, reaching mean targeted integration values of 21% and 16% respectively, that were associated with the phenotypic correction of these cells. Overall, our results demonstrate the feasibility of implementing a therapeutic targeted integration strategy into the mMbs85 locus, ortholog to the well-validated hAAVS1, constituting the first study of gene editing in mHSC with TALEN, that sets the basis for the use of a new safe harbor locus in mice.
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http://dx.doi.org/10.1038/s41598-020-63971-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181878PMC
April 2020

Mosaicism in Fanconi anemia: concise review and evaluation of published cases with focus on clinical course of blood count normalization.

Ann Hematol 2020 May 17;99(5):913-924. Epub 2020 Feb 17.

Rocket Pharmaceuticals, Inc., New York, NY, USA.

Fanconi anemia (FA) is a DNA repair disorder resulting from mutations in genes encoding for FA DNA repair complex components and is characterized by variable congenital abnormalities, bone marrow failure (BMF), and high incidences of malignancies. FA mosaicism arises from reversion or other compensatory mutations in hematopoietic cells and may be associated with BMF reversal and decreased blood cell sensitivity to DNA-damaging agents (clastogens); this sensitivity is a phenotypic and diagnostic hallmark of FA. Uncertainty regarding the clinical significance of FA mosaicism persists; in some cases, patients have survived multiple decades without BMF or hematologic malignancy, and in others hematologic failure occurred despite the presence of clastogen-resistant cell populations. Assessment of mosaicism is further complicated because clinical evaluation is frequently based on clastogen resistance in lymphocytes, which may arise from reversion events both in lymphoid-specific lineages and in more pluripotent hematopoietic stem/progenitor cells (HSPCs). In this review, we describe diagnostic methods and outcomes in published mosaicism series, including the substantial intervals (1-6 years) over which blood counts normalized, and the relatively favorable clinical course in cases where clastogen resistance was demonstrated in bone marrow progenitors. We also analyzed published FA mosaic cases with emphasis on long-term clinical outcomes when blood count normalization was identified. Blood count normalization in FA mosaicism likely arises from reversion events in long-term primitive HSPCs and is associated with low incidences of BMF or hematologic malignancy. These observations have ramifications for current investigational therapeutic programs in FA intended to enable gene correction in long-term repopulating HSPCs.
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http://dx.doi.org/10.1007/s00277-020-03954-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196946PMC
May 2020

Humanized mice are precious tools for evaluation of hematopoietic gene therapies and preclinical modeling to move towards a clinical trial.

Biochem Pharmacol 2020 04 11;174:113711. Epub 2019 Nov 11.

CIRI, Université de Lyon, INSERM U1111, ENS de Lyon, Université Lyon1, CNRS, UMR 5308, 69007 Lyon, France; Université Côte d'Azur, INSERM, C3M, 06204 Nice, France. Electronic address:

Over the last decade, incrementally improved xenograft mouse models, which support the engraftment and development of a human hemato-lymphoid system, have been developed and represent an important fundamental and preclinical research tool. Immunodeficient mice can be transplanted with human hematopoietic stem cells (HSCs) and this process is accompanied by HSC homing to the murine bone marrow. This is followed by stem cell expansion, multilineage hematopoiesis, long-term engraftment, and functional human antibody and cellular immune responses. The most significant contributions made by these humanized mice are the identification of normal and leukemic hematopoietic stem cells, the characterization of the human hematopoietic hierarchy, screening of anti-cancer therapies and their use as preclinical models for gene therapy applications. This review article focuses on several gene therapy applications that have benefited from evaluation in humanized mice such as chimeric antigen receptor (CAR) T cell therapies for cancer, anti-viral therapies and gene therapies for multiple monogenetic diseases. Humanized mouse models have been and still are of great value for the gene therapy field since they provide a more reliable understanding of sometimes complicated therapeutic approaches such as recently developed therapeutic gene editing strategies, which seek to correct a gene at its endogenous genomic locus. Additionally, humanized mouse models, which are of great importance with regard to testing new vector technologies in vivo for assessing safety and efficacy prior toclinical trials, help to expedite the critical translation from basic findings to clinical applications. In this review, innovative gene therapies and preclinical studies to evaluate T- and B-cell and HSC-based therapies in humanized mice are discussed and illustrated by multiple examples.
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http://dx.doi.org/10.1016/j.bcp.2019.113711DOI Listing
April 2020

Optimised molecular genetic diagnostics of Fanconi anaemia by whole exome sequencing and functional studies.

J Med Genet 2020 04 5;57(4):258-268. Epub 2019 Oct 5.

Hospital Universitario Puerta del Mar, Cadiz, Andalucía, Spain.

Purpose: Patients with Fanconi anaemia (FA), a rare DNA repair genetic disease, exhibit chromosome fragility, bone marrow failure, malformations and cancer susceptibility. FA molecular diagnosis is challenging since FA is caused by point mutations and large deletions in 22 genes following three heritability patterns. To optimise FA patients' characterisation, we developed a simplified but effective methodology based on whole exome sequencing (WES) and functional studies.

Methods: 68 patients with FA were analysed by commercial WES services. Copy number variations were evaluated by sequencing data analysis with RStudio. To test missense variants, wt FANCA cDNA was cloned and variants were introduced by site-directed mutagenesis. Vectors were then tested for their ability to complement DNA repair defects of a FANCA-KO human cell line generated by TALEN technologies.

Results: We identified 93.3% of mutated alleles including large deletions. We determined the pathogenicity of three FANCA missense variants and demonstrated that two variants reported in mutations databases as 'affecting functions' are SNPs. Deep analysis of sequencing data revealed patients' true mutations, highlighting the importance of functional analysis. In one patient, no pathogenic variant could be identified in any of the 22 known FA genes, and in seven patients, only one deleterious variant could be identified (three patients each with FANCA and FANCD2 and one patient with FANCE mutations) CONCLUSION: WES and proper bioinformatics analysis are sufficient to effectively characterise patients with FA regardless of the rarity of their complementation group, type of mutations, mosaic condition and DNA source.
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http://dx.doi.org/10.1136/jmedgenet-2019-106249DOI Listing
April 2020

NHEJ-Mediated Repair of CRISPR-Cas9-Induced DNA Breaks Efficiently Corrects Mutations in HSPCs from Patients with Fanconi Anemia.

Cell Stem Cell 2019 11 19;25(5):607-621.e7. Epub 2019 Sep 19.

Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT), Madrid 28040, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER-ISCIII), Madrid 28040, Spain; Advanced Therapies Unit, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD/UAM), Madrid 28040, Spain. Electronic address:

Non-homologous end-joining (NHEJ) is the preferred mechanism used by hematopoietic stem cells (HSCs) to repair double-stranded DNA breaks and is particularly increased in cells deficient in the Fanconi anemia (FA) pathway. Here, we show feasible correction of compromised functional phenotypes in hematopoietic cells from multiple FA complementation groups, including FA-A, FA-C, FA-D1, and FA-D2. NHEJ-mediated repair of targeted CRISPR-Cas9-induced DNA breaks generated compensatory insertions and deletions that restore the coding frame of the mutated gene. NHEJ-mediated editing efficacy was initially verified in FA lymphoblastic cell lines and then in primary FA patient-derived CD34 cells, which showed marked proliferative advantage and phenotypic correction both in vitro and after transplantation. Importantly, and in contrast to homologous directed repair, NHEJ efficiently targeted primitive human HSCs, indicating that NHEJ editing approaches may constitute a sound alternative for editing self-renewing human HSCs and consequently for treatment of FA and other monogenic diseases affecting the hematopoietic system.
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http://dx.doi.org/10.1016/j.stem.2019.08.016DOI Listing
November 2019

Successful engraftment of gene-corrected hematopoietic stem cells in non-conditioned patients with Fanconi anemia.

Nat Med 2019 09 9;25(9):1396-1401. Epub 2019 Sep 9.

Centro de Investigación Biomédica en Red de Enfermedades Raras, Madrid, Spain.

Fanconi anemia (FA) is a DNA repair syndrome generated by mutations in any of the 22 FA genes discovered to date. Mutations in FANCA account for more than 60% of FA cases worldwide. Clinically, FA is associated with congenital abnormalities and cancer predisposition. However, bone marrow failure is the primary pathological feature of FA that becomes evident in 70-80% of patients with FA during the first decade of life. In this clinical study (ClinicalTrials.gov, NCT03157804 ; European Clinical Trials Database, 2011-006100-12), we demonstrate that lentiviral-mediated hematopoietic gene therapy reproducibly confers engraftment and proliferation advantages of gene-corrected hematopoietic stem cells (HSCs) in non-conditioned patients with FA subtype A. Insertion-site analyses revealed the multipotent nature of corrected HSCs and showed that the repopulation advantage of these cells was not due to genotoxic integrations of the therapeutic provirus. Phenotypic correction of blood and bone marrow cells was shown by the acquired resistance of hematopoietic progenitors and T lymphocytes to DNA cross-linking agents. Additionally, an arrest of bone marrow failure progression was observed in patients with the highest levels of gene marking. The progressive engraftment of corrected HSCs in non-conditioned patients with FA supports that gene therapy should constitute an innovative low-toxicity therapeutic option for this life-threatening disorder.
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http://dx.doi.org/10.1038/s41591-019-0550-zDOI Listing
September 2019

Advances in the gene therapy of monogenic blood cell diseases.

Clin Genet 2020 01 11;97(1):89-102. Epub 2019 Jul 11.

Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Madrid, Spain.

Hematopoietic gene therapy has markedly progressed during the last 15 years both in terms of safety and efficacy. While a number of serious adverse events (SAE) were initially generated as a consequence of genotoxic insertions of gamma-retroviral vectors in the cell genome, no SAEs and excellent outcomes have been reported in patients infused with autologous hematopoietic stem cells (HSCs) transduced with self-inactivated lentiviral and gammaretroviral vectors. Advances in the field of HSC gene therapy have extended the number of monogenic diseases that can be treated with these approaches. Nowadays, evidence of clinical efficacy has been shown not only in primary immunodeficiencies, but also in other hematopoietic diseases, including beta-thalassemia and sickle cell anemia. In addition to the rapid progression of non-targeted gene therapies in the clinic, new approaches based on gene editing have been developed thanks to the discovery of designed nucleases and improved non-integrative vectors, which have markedly increased the efficacy and specificity of gene targeting to levels compatible with its clinical application. Based on advances achieved in the field of gene therapy, it can be envisaged that these therapies will soon be part of the therapeutic approaches used to treat life-threatening diseases of the hematopoietic system.
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http://dx.doi.org/10.1111/cge.13593DOI Listing
January 2020

Advances in Gene Therapy for Fanconi Anemia.

Hum Gene Ther 2018 10;29(10):1114-1123

1 Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Madrid, Spain; Madrid, Spain .

Fanconi anemia (FA) is a rare inherited disease that is associated with bone marrow failure and a predisposition to cancer. Previous clinical trials emphasized the difficulties that accompany the use of gene therapy to treat bone marrow failure in patients with FA. Nevertheless, the discovery of new drugs that can efficiently mobilize hematopoietic stem cells (HSCs) and the development of optimized procedures for transducing HSCs, using safe, integrative vectors, markedly improved the efficiency by which the phenotype of hematopoietic repopulating cells from patients with FA can be corrected. In addition, these achievements allowed the demonstration of the in vivo proliferation advantage of gene-corrected FA repopulating cells in immunodeficient mice. Significantly, new gene therapy trials are currently ongoing to investigate the progressive restoration of hematopoiesis in patients with FA by gene-corrected autologous HSCs. Further experimental studies are focused on the ex vivo transduction of unpurified FA HSCs, using new pseudotyped vectors that have HSC tropism. Because of the resistance of some of these vectors to serum complement, new strategies for in vivo gene therapy for FA HSCs are in development. Finally, because of the rapid advancements in gene-editing techniques, correction of CD34 cells isolated from patients with FA is now feasible, using gene-targeting strategies. Taken together, these advances indicate that gene therapy can soon be used as an efficient and safe alternative for the hematopoietic treatment of patients with FA.
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http://dx.doi.org/10.1089/hum.2018.124DOI Listing
October 2018

Measles virus envelope pseudotyped lentiviral vectors transduce quiescent human HSCs at an efficiency without precedent.

Blood Adv 2017 Oct 24;1(23):2088-2104. Epub 2017 Oct 24.

International Center for Infectiology Research, Team Enveloped Viruses, Vectors and Innate Responses, INSERM, U1111, Université Claude Bernard Lyon 1, Centre National de la Recherche Scientifique, Unité Mixte de Recherche (UMR) 5308, Ecole Normale Supérieure de Lyon, Lyon, France.

Hematopoietic stem cell (HSC)-based gene therapy trials are now moving toward the use of lentiviral vectors (LVs) with success. However, one challenge in the field remains: efficient transduction of HSCs without compromising their stem cell potential. Here we showed that measles virus glycoprotein-displaying LVs (hemagglutinin and fusion protein LVs [H/F-LVs]) were capable of transducing 100% of early-acting cytokine-stimulated human CD34 (hCD34) progenitor cells upon a single application. Strikingly, these H/F-LVs also allowed transduction of up to 70% of nonstimulated quiescent hCD34 cells, whereas conventional vesicular stomatitis virus G (VSV-G)-LVs reached 5% at the most with H/F-LV entry occurring exclusively through the CD46 complement receptor. Importantly, reconstitution of NOD/SCIDγc (NSG) mice with H/F-LV transduced prestimulated or resting hCD34 cells confirmed these high transduction levels in all myeloid and lymphoid lineages. Remarkably, for resting CD34 cells, secondary recipients exhibited increasing transduction levels of up to 100%, emphasizing that H/F-LVs efficiently gene-marked HSCs in the resting state. Because H/F-LVs promoted ex vivo gene modification of minimally manipulated CD34 progenitors that maintained stemness, we assessed their applicability in Fanconi anemia, a bone marrow (BM) failure with chromosomal fragility. Notably, only H/F-LVs efficiently gene-corrected minimally stimulated hCD34 cells in unfractionated BM from these patients. These H/F-LVs improved HSC gene delivery in the absence of cytokine stimulation while maintaining their stem cell potential. Thus, H/F-LVs will facilitate future clinical applications requiring HSC gene modification, including BM failure syndromes, for which treatment has been very challenging up to now.
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http://dx.doi.org/10.1182/bloodadvances.2017007773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728281PMC
October 2017

Therapeutic gene editing in CD34 hematopoietic progenitors from Fanconi anemia patients.

EMBO Mol Med 2017 11;9(11):1574-1588

Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain

Gene targeting constitutes a new step in the development of gene therapy for inherited diseases. Although previous studies have shown the feasibility of editing fibroblasts from Fanconi anemia (FA) patients, here we aimed at conducting therapeutic gene editing in clinically relevant cells, such as hematopoietic stem cells (HSCs). In our first experiments, we showed that zinc finger nuclease (ZFN)-mediated insertion of a non-therapeutic EGFP-reporter donor in the "safe harbor" locus of FA-A lymphoblastic cell lines (LCLs), indicating that FANCA is not essential for the editing of human cells. When the same approach was conducted with therapeutic donors, an efficient phenotypic correction of FA-A LCLs was obtained. Using primary cord blood CD34 cells from healthy donors, gene targeting was confirmed not only in cultured cells, but also in hematopoietic precursors responsible for the repopulation of primary and secondary immunodeficient mice. Moreover, when similar experiments were conducted with mobilized peripheral blood CD34 cells from FA-A patients, we could demonstrate for the first time that gene targeting in primary hematopoietic precursors from FA patients is feasible and compatible with the phenotypic correction of these clinically relevant cells.
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http://dx.doi.org/10.15252/emmm.201707540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666315PMC
November 2017

Engraftment and in vivo proliferation advantage of gene-corrected mobilized CD34 cells from Fanconi anemia patients.

Blood 2017 09 11;130(13):1535-1542. Epub 2017 Aug 11.

Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain.

Previous Fanconi anemia (FA) gene therapy studies have failed to demonstrate engraftment of gene-corrected hematopoietic stem and progenitor cells (HSPCs) from FA patients, either after autologous transplantation or infusion into immunodeficient mice. In this study, we demonstrate that a validated short transduction protocol of G-CSF plus plerixafor-mobilized CD34 cells from FA-A patients with a therapeutic lentiviral vector corrects the phenotype of in vitro cultured hematopoietic progenitor cells. Transplantation of transduced FA CD34 cells into immunodeficient mice resulted in reproducible engraftment of myeloid, lymphoid, and CD34 cells. Importantly, a marked increase in the proportion of phenotypically corrected, patient-derived hematopoietic cells was observed after transplantation with respect to the infused CD34 graft, indicating the proliferative advantage of corrected FA-A hematopoietic repopulating cells. Our data demonstrate for the first time that optimized protocols of hematopoietic stem cell collection from FA patients, followed by the short and clinically validated transduction of these cells with a therapeutic lentiviral vector, results in the generation of phenotypically corrected HSPCs capable of repopulating and developing proliferation advantage in immunodeficient mice. Our results suggest that clinical approaches for FA gene therapy similar to those used in this study will facilitate hematopoietic repopulation in FA patients with gene corrected HSPCs, opening new prospects for gene therapy of FA patients.
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http://dx.doi.org/10.1182/blood-2017-03-774174DOI Listing
September 2017

Gene Therapy in Fanconi Anemia: A Matter of Time, Safety and Gene Transfer Tool Efficiency.

Curr Gene Ther 2017 ;16(5):297-308

CIEMAT. Avda. Complutense 40, 28040 Madrid, Spain.

Fanconi anemia (FA) is a rare genetic syndrome characterized by progressive marrow failure. Gene therapy by infusion of FA-corrected autologous hematopoietic stem cells (HSCs) may offer a potential cure since it is a monogenetic disease with mutations in the FANC genes, coding for DNA repair enzymes [1]. However, the collection of hCD34+-cells in FA patients implies particular challenges because of the reduced numbers of progenitor cells present in their bone marrow (BM) [2] or mobilized peripheral blood [3-5]. In addition, the FA genetic defect fragilizes the HSCs [6]. These particular features might explain why the first clinical trials using murine leukemia virus derived retroviral vectors conducted for FA failed to show engraftment of corrected cells. The gene therapy field is now moving towards the use of lentiviral vectors (LVs) evidenced by recent succesful clinical trials for the treatment of patients suffering from adrenoleukodystrophy (ALD) [7], β-thalassemia [8], metachromatic leukodystrophy [9] and Wiskott-Aldrich syndrome [10]. LV trials for X-linked severe combined immunodificiency and Fanconi anemia (FA) defects were recently initiated [11, 12]. Fifteen years of preclinical studies using different FA mouse models and in vitro research allowed us to find the weak points in the in vitro culture and transduction conditions, which most probably led to the initial failure of FA HSC gene therapy. In this review, we will focus on the different obstacles, unique to FA gene therapy, and how they have been overcome through the development of optimized protocols for FA HSC culture and transduction and the engineering of new gene transfer tools for FA HSCs. These combined advances in the field hopefully will allow the correction of the FA hematological defect in the near future.
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http://dx.doi.org/10.2174/1566523217666170109114309DOI Listing
July 2018

TGF-β: a master regulator of the bone marrow failure puzzle in Fanconi anemia.

Stem Cell Investig 2016 7;3:75. Epub 2016 Nov 7.

Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER-ISCIII), Madrid, Spain;; Instituto de Investigación Sanitaria de la Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain.

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http://dx.doi.org/10.21037/sci.2016.09.17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5104581PMC
November 2016

Direct Conversion of Fibroblasts to Megakaryocyte Progenitors.

Cell Rep 2016 10;17(3):671-683

Center of Regenerative Medicine in Barcelona (CMRB), Barcelona Biomedical Research Park, Dr. Aiguader 88, 08003 Barcelona, Spain; Center for Networked Biomedical Research on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 28029 Madrid, Spain; Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain. Electronic address:

Current sources of platelets for transfusion are insufficient and associated with risk of alloimmunization and blood-borne infection. These limitations could be addressed by the generation of autologous megakaryocytes (MKs) derived in vitro from somatic cells with the ability to engraft and differentiate in vivo. Here, we show that overexpression of a defined set of six transcription factors efficiently converts mouse and human fibroblasts into MK-like progenitors. The transdifferentiated cells are CD41, display polylobulated nuclei, have ploidies higher than 4N, form MK colonies, and give rise to platelets in vitro. Moreover, transplantation of MK-like murine progenitor cells into NSG mice results in successful engraftment and further maturation in vivo. Similar results are obtained using disease-corrected fibroblasts from Fanconi anemia patients. Our results combined demonstrate that functional MK progenitors with clinical potential can be obtained in vitro, circumventing the use of hematopoietic progenitors or pluripotent stem cells.
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http://dx.doi.org/10.1016/j.celrep.2016.09.036DOI Listing
October 2016

BCR-JAK2 drives a myeloproliferative neoplasm in transplanted mice.

J Pathol 2015 Jun 3;236(2):219-28. Epub 2015 Mar 3.

Molecular Biology Unit, Instituto de Investigación Sanitaria Princesa (IIS-P, UAM), Hospital Universitario de La Princesa, Madrid, Spain.

BCR-JAK2 is an infrequent gene fusion found in chronic/acute, myeloid/lymphoid Philadelphia chromosome-negative leukaemia. In this study, we demonstrated that in vivo expression of BCR-JAK2 in mice induces neoplasia, with fatal consequences. Transplantation of BCR-JAK2 bone marrow progenitors promoted splenomegaly, with megakaryocyte infiltration and elevated leukocytosis of myeloid origin. Analysis of peripheral blood revealed the presence of immature myeloid cells, platelet aggregates and ineffective erythropoiesis. A possible molecular mechanism for these observations involved inhibition of apoptosis by deregulated expression of the anti-apoptotic mediator Bcl-xL and the serine/threonine kinase Pim1. Together, these data provide a suitable in vivo molecular mechanism for leukaemia induction by BCR-JAK2 that validates the use of this model as a relevant preclinical tool for the design of new targeted therapies in Philadelphia chromosome-negative leukaemia involving BCR-JAK2-driven activation of the JAK2 pathway.
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http://dx.doi.org/10.1002/path.4513DOI Listing
June 2015

Targeted gene therapy and cell reprogramming in Fanconi anemia.

EMBO Mol Med 2014 Jun 6;6(6):835-48. Epub 2014 Apr 6.

Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain

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.
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http://dx.doi.org/10.15252/emmm.201303374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4203359PMC
June 2014

Mutations in ERCC4, encoding the DNA-repair endonuclease XPF, cause Fanconi anemia.

Am J Hum Genet 2013 May 25;92(5):800-6. Epub 2013 Apr 25.

Genome Instability and DNA Repair Group, Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain.

Fanconi anemia (FA) is a rare genomic instability disorder characterized by progressive bone marrow failure and predisposition to cancer. FA-associated gene products are involved in the repair of DNA interstrand crosslinks (ICLs). Fifteen FA-associated genes have been identified, but the genetic basis in some individuals still remains unresolved. Here, we used whole-exome and Sanger sequencing on DNA of unclassified FA individuals and discovered biallelic germline mutations in ERCC4 (XPF), a structure-specific nuclease-encoding gene previously connected to xeroderma pigmentosum and segmental XFE progeroid syndrome. Genetic reversion and wild-type ERCC4 cDNA complemented the phenotype of the FA cell lines, providing genetic evidence that mutations in ERCC4 cause this FA subtype. Further biochemical and functional analysis demonstrated that the identified FA-causing ERCC4 mutations strongly disrupt the function of XPF in DNA ICL repair without severely compromising nucleotide excision repair. Our data show that depending on the type of ERCC4 mutation and the resulting balance between both DNA repair activities, individuals present with one of the three clinically distinct disorders, highlighting the multifunctional nature of the XPF endonuclease in genome stability and human disease.
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http://dx.doi.org/10.1016/j.ajhg.2013.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3644630PMC
May 2013

Transforming and tumorigenic activity of JAK2 by fusion to BCR: molecular mechanisms of action of a novel BCR-JAK2 tyrosine-kinase.

PLoS One 2012 27;7(2):e32451. Epub 2012 Feb 27.

Molecular Biology Unit, Hospital Universitario de la Princesa, Instituto de Investigación Sanitaria Princesa and Red Española de Investigación en Patología Infecciosa, Madrid, Spain.

Chromosomal translocations in tumors frequently produce fusion genes coding for chimeric proteins with a key role in oncogenesis. Recent reports described a BCR-JAK2 fusion gene in fatal chronic and acute myeloid leukemia, but the functional behavior of the chimeric protein remains uncharacterized. We used fluorescence in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR) assays to describe a BCR-JAK2 fusion gene from a patient with acute lymphoblastic leukemia. The patient has been in complete remission for six years following treatment and autologous transplantation, and minimal residual disease was monitored by real-time RT-PCR. BCR-JAK2 codes for a protein containing the BCR oligomerization domain fused to the JAK2 tyrosine-kinase domain. In vitro analysis of transfected cells showed that BCR-JAK2 is located in the cytoplasm. Transduction of hematopoietic Ba/F3 cells with retroviral vectors carrying BCR-JAK2 induced IL-3-independent cell growth, constitutive activation of the chimeric protein as well as STAT5 phosphorylation and translocation to the nuclei, where Bcl-xL gene expression was elicited. Primary mouse progenitor cells transduced with BCR-JAK2 also showed increased proliferation and survival. Treatment with the JAK2 inhibitor TG101209 abrogated BCR-JAK2 and STAT5 phosphorylation, decreased Bcl-xL expression and triggered apoptosis of transformed Ba/F3 cells. Therefore, BCR-JAK2 is a novel tyrosine-kinase with transforming activity. It deregulates growth factor-dependent proliferation and cell survival, which can be abrogated by the TG101209 inhibitor. Moreover, transformed Ba/F3 cells developed tumors when injected subcutaneously into nude mice, thus proving the tumorigenic capacity of BCR-JAK2 in vivo. Together these findings suggest that adult and pediatric patients with BCR-ABL-negative leukemia and JAK2 overexpression may benefit from targeted therapies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0032451PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3288102PMC
August 2012

Down-regulated expression of hsa-miR-181c in Fanconi anemia patients: implications in TNFα regulation and proliferation of hematopoietic progenitor cells.

Blood 2012 Mar 6;119(13):3042-9. Epub 2012 Feb 6.

Hematopoiesis and Gene Therapy Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain.

Fanconi anemia (FA) is an inherited genetic disorder associated with BM failure and cancer predisposition. In the present study, we sought to elucidate the role of microRNAs (miRNAs) in the hematopoietic defects observed in FA patients. Initial studies showed that 3 miRNAs, hsa-miR-133a, hsa-miR-135b, and hsa-miR-181c, were significantly down-regulated in lymphoblastoid cell lines and fresh peripheral blood cells from FA patients. In vitro studies with cells expressing the luciferase reporter fused to the TNFα 3'-untranslated region confirmed in silico predictions suggesting an interaction between hsa-miR-181c and TNFα mRNA. These observations were consistent with the down-regulated expression of TNFα mediated by hsa-miR-181c in cells from healthy donors and cells from FA patients. Because of the relevance of TNFα in the hematopoietic defects of FA patients, in the present study, we transfected BM cells from FA patients with hsa-miR-181c to evaluate the impact of this miRNA on their clonogenic potential. hsa-miR-181c markedly increased the number and size of the myeloid and erythroid colonies generated by BM cells from FA patients. Our results offer new clues toward understanding the biologic basis of BM failure in FA patients and open new possibilities for the treatment of the hematologic dysfunction in FA patients based on miRNA regulation.
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http://dx.doi.org/10.1182/blood-2011-01-331017DOI Listing
March 2012

A novel lentiviral vector targets gene transfer into human hematopoietic stem cells in marrow from patients with bone marrow failure syndrome and in vivo in humanized mice.

Blood 2012 Feb 23;119(5):1139-50. Epub 2011 Nov 23.

INSERM U758, Human Virology Department, Enveloppe Virale and Ingeniérie du Retrovirus, Ecole Nomrale Supérieure de Lyon, and Université de Lyon, 46 Allée d'Italie, Lyon Cedex 07, France.

In vivo lentiviral vector (LV)-mediated gene delivery would represent a great step forward in the field of gene therapy. Therefore, we have engineered a novel LV displaying SCF and a mutant cat endogenous retroviral glycoprotein, RDTR. These RDTR/SCF-LVs outperformed RDTR-LVs for transduction of human CD34(+) cells (hCD34(+)). For in vivo gene therapy, these novel RDTR/SCF-displaying LVs can distinguish between the target hCD34(+) cells of interest and nontarget cells. Indeed, they selectively targeted transduction to 30%-40% of the hCD34(+) cells in cord blood mononuclear cells and in the unfractionated BM of healthy and Fanconi anemia donors, resulting in the correction of CD34(+) cells in the patients. Moreover, RDTR/SCF-LVs targeted transduction to CD34(+) cells with 95-fold selectivity compared with T cells in total cord blood. Remarkably, in vivo injection of the RDTR/SCF-LVs into the BM cavity of humanized mice resulted in the highly selective transduction of candidate hCD34(+)Lin(-) HSCs. In conclusion, this new LV will facilitate HSC-based gene therapy by directly targeting these primitive cells in BM aspirates or total cord blood. Most importantly, in the future, RDTR/SCF-LVs might completely obviate ex vivo handling and simplify gene therapy for many hematopoietic defects because of their applicability to direct in vivo inoculation.
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http://dx.doi.org/10.1182/blood-2011-04-346619DOI Listing
February 2012

Stem cell gene therapy for fanconi anemia: report from the 1st international Fanconi anemia gene therapy working group meeting.

Mol Ther 2011 Jul 3;19(7):1193-8. Epub 2011 May 3.

Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota 55455, USA.

Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA.
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http://dx.doi.org/10.1038/mt.2011.78DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3129570PMC
July 2011

Bcr/Abl interferes with the Fanconi anemia/BRCA pathway: implications in the chromosomal instability of chronic myeloid leukemia cells.

PLoS One 2010 Dec 28;5(12):e15525. Epub 2010 Dec 28.

Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas and Centro de Investigación Biomédica en Red de Enfermedades Raras, Madrid, Spain.

Chronic myeloid leukemia (CML) is a malignant clonal disorder of the hematopoietic system caused by the expression of the BCR/ABL fusion oncogene. Although it is well known that CML cells are genetically unstable, the mechanisms accounting for this genomic instability are still poorly understood. Because the Fanconi anemia (FA) pathway is believed to control several mechanisms of DNA repair, we investigated whether this pathway was disrupted in CML cells. Our data show that CML cells have a defective capacity to generate FANCD2 nuclear foci, either in dividing cells or after DNA damage. Similarly, human cord blood CD34(+) cells transduced with BCR/ABL retroviral vectors showed impaired FANCD2 foci formation, whereas FANCD2 monoubiquitination in these cells was unaffected. Soon after the transduction of CD34(+) cells with BCR/ABL retroviral vectors a high proportion of cells with supernumerary centrosomes was observed. Similarly, BCR/ABL induced a high proportion of chromosomal abnormalities, while mediated a cell survival advantage after exposure to DNA cross-linking agents. Significantly, both the impaired formation of FANCD2 nuclear foci, and also the predisposition of BCR/ABL cells to develop centrosomal and chromosomal aberrations were reverted by the ectopic expression of BRCA1. Taken together, our data show for the first time a disruption of the FA/BRCA pathway in BCR/ABL cells, suggesting that this defective pathway should play an important role in the genomic instability of CML by the co-occurrence of centrosomal amplification and DNA repair deficiencies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015525PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3011007PMC
December 2010

Development of lentiviral vectors with optimized transcriptional activity for the gene therapy of patients with Fanconi anemia.

Hum Gene Ther 2010 May;21(5):623-30

Division of Hematopoiesis and Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales, y Tecnológicas (CIEMAT), and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBER-ER), 28040 Madrid, Spain.

Fanconi anemia (FA) is an inherited genetic disease characterized mainly by bone marrow failure and cancer predisposition. Although gene therapy may constitute a good therapeutic option for many patients with FA, none of the clinical trials so far developed has improved the clinical status of these patients. We have proposed strategies for the genetic correction of bone marrow grafts from patients with FA, using lentiviral vectors (LVs). Here we investigate the relevance of the expression of FANCA to confer a therapeutic effect in cells from patients with FA-A, the most frequent complementation group in FA. Our data show that relatively weak promoters such as the vav or phosphoglycerate kinase (PGK) promoter confer, per copy of FANCA, physiological levels of FANCA mRNA in lymphoblastoid cell lines, whereas the cytomegalovirus and, more significantly, spleen focus-forming virus (SFFV) promoters mediated the expression of supraphysiological levels of FANCA mRNA. Insertion of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or a mutated WPRE into the 3' region of PGK-FANCA LVs significantly increased FANCA mRNA levels. At the protein level, however, all tested vectors conferred, per copy of FANCA, similar and physiological levels of the protein, except SFFV LVs, which again conferred supraphysiological levels of FANCA. In spite of their different activity, all tested vectors mediated a similar phenotypic correction in FA-A lymphoblastoid cell lines and also in hematopoietic progenitors from patients with FA-A. On the basis of the efficacy and safety properties of PGK LVs, a PGK LV carrying FANCA and a mutant WPRE is proposed as an optimized vector for the gene therapy of patients with FA-A.
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http://dx.doi.org/10.1089/hum.2009.141DOI Listing
May 2010

Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells.

Nature 2009 Jul 31;460(7251):53-9. Epub 2009 May 31.

Center for Regenerative Medicine in Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain.

The generation of induced pluripotent stem (iPS) cells has enabled the derivation of patient-specific pluripotent cells and provided valuable experimental platforms to model human disease. Patient-specific iPS cells are also thought to hold great therapeutic potential, although direct evidence for this is still lacking. Here we show that, on correction of the genetic defect, somatic cells from Fanconi anaemia patients can be reprogrammed to pluripotency to generate patient-specific iPS cells. These cell lines appear indistinguishable from human embryonic stem cells and iPS cells from healthy individuals. Most importantly, we show that corrected Fanconi-anaemia-specific iPS cells can give rise to haematopoietic progenitors of the myeloid and erythroid lineages that are phenotypically normal, that is, disease-free. These data offer proof-of-concept that iPS cell technology can be used for the generation of disease-corrected, patient-specific cells with potential value for cell therapy applications.
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http://dx.doi.org/10.1038/nature08129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2720823PMC
July 2009

Elevated levels of IL-1beta in Fanconi anaemia group A patients due to a constitutively active phosphoinositide 3-kinase-Akt pathway are capable of promoting tumour cell proliferation.

Biochem J 2009 Jul 29;422(1):161-70. Epub 2009 Jul 29.

Unidad de Genética Molecular, Hospital Universitario Marqués de Valdecilla, Santander, Spain.

FA (Fanconi anaemia) is a hereditary disease characterized by congenital malformations, progressive bone marrow failure and an extraordinary elevated predisposition to develop cancer. In the present manuscript we describe an anomalous high level of the proinflammatory cytokine IL-1beta (interleukin-1beta) present in the serum of FA patients. The elevated levels of IL-1beta were completely reverted by transduction of a wild-type copy of the FancA cDNA into FA-A (FA group A) lymphocytes. Although the transcription factor NF-kappaB (nuclear factor-kappaB) is a well established regulator of IL-1beta expression, our experiments did not show any proof of elevated NF-kappaB activity in FA-A cells. However, we found that the overexpression of IL-1beta in FA-A cells is related to a constitutively activated PI3K (phosphoinositide 3-kinase)-Akt pathway in these cells. We provide evidence that the effect of Akt on IL-1beta activation is mediated by the inhibition of GSK3beta (glycogen synthase kinase 3beta). Finally, our data indicate that the levels of IL-1beta produced by FA-A lymphoblasts are enough to promote an activation of the cell cycle in primary glioblastoma progenitor cells. Together, these results demonstrate that the constitutive activation of the PI3K-Akt pathway in FA cells upregulates the expression of IL-1beta through an NF-kappaB-independent mechanism and that this overproduction activates the proliferation of tumour cells.
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http://dx.doi.org/10.1042/BJ20082118DOI Listing
July 2009

Lentiviral-mediated genetic correction of hematopoietic and mesenchymal progenitor cells from Fanconi anemia patients.

Mol Ther 2009 Jun 10;17(6):1083-92. Epub 2009 Mar 10.

División de Hematopoyesis y Terapia Génica, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain.

Previous clinical trials based on the genetic correction of purified CD34(+) cells with gamma-retroviral vectors have demonstrated clinical efficacy in different monogenic diseases, including X-linked severe combined immunodeficiency, adenosine deaminase deficient severe combined immunodeficiency and chronic granulomatous disease. Similar protocols, however, failed to engraft Fanconi anemia (FA) patients with genetically corrected cells. In this study, we first aimed to correlate the hematological status of 27 FA patients with CD34(+) cell values determined in their bone marrow (BM). Strikingly, no correlation between these parameters was observed, although good correlations were obtained when numbers of colony-forming cells (CFCs) were considered. Based on these results, and because purified FA CD34(+) cells might have suboptimal repopulating properties, we investigated the possibility of genetically correcting unselected BM samples from FA patients. Our data show that the lentiviral transduction of unselected FA BM cells mediates an efficient phenotypic correction of hematopoietic progenitor cells and also of CD34(-) mesenchymal stromal cells (MSCs), with a reported role in hematopoietic engraftment. Our results suggest that gene therapy protocols appropriate for the treatment of different monogenic diseases may not be adequate for stem cell diseases like FA. We propose a new approach for the gene therapy of FA based on the rapid transduction of unselected hematopoietic grafts with lentiviral vectors (LVs).
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http://dx.doi.org/10.1038/mt.2009.26DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2835196PMC
June 2009