RESUMEN
Idiopathic pulmonary fibrosis is a lethal lung fibrotic disease, associated with aging with a mean survival of 2-5 years and no curative treatment. The GSE4 peptide is able to rescue cells from senescence, DNA and oxidative damage, inflammation, and induces telomerase activity. Here, we investigated the protective effect of GSE4 expression in vitro in rat alveolar epithelial cells (AECs), and in vivo in a bleomycin model of lung fibrosis. Bleomycin-injured rat AECs, expressing GSE4 or treated with GSE4-PLGA/PEI nanoparticles showed an increase of telomerase activity, decreased DNA damage, and decreased expression of IL6 and cleaved-caspase 3. In addition, these cells showed an inhibition in expression of fibrotic markers induced by TGF-ß such as collagen-I and III among others. Furthermore, treatment with GSE4-PLGA/PEI nanoparticles in a rat model of bleomycin-induced fibrosis, increased telomerase activity and decreased DNA damage in proSP-C cells. Both in preventive and therapeutic protocols GSE4-PLGA/PEI nanoparticles prevented and attenuated lung damage monitored by SPECT-CT and inhibited collagen deposition. Lungs of rats treated with bleomycin and GSE4-PLGA/PEI nanoparticles showed reduced expression of α-SMA and pro-inflammatory cytokines, increased number of pro-SPC-multicellular structures and increased DNA synthesis in proSP-C cells, indicating therapeutic efficacy of GSE4-nanoparticles in experimental lung fibrosis and a possible curative treatment for lung fibrotic patients.
Asunto(s)
Apoptosis/efectos de los fármacos , Bleomicina/farmacología , Daño del ADN/efectos de los fármacos , Pulmón/efectos de los fármacos , Nanopartículas/uso terapéutico , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/metabolismo , Colágeno/efectos de los fármacos , Colágeno/metabolismo , Humanos , Pulmón/metabolismo , Estrés Oxidativo/efectos de los fármacos , Péptidos/farmacologíaRESUMEN
Dyskeratosis congenita (DC) is a rare telomere biology disorder, which results in different clinical manifestations, including severe bone marrow failure. To date, the only curative treatment for the bone marrow failure in DC patients is allogeneic hematopoietic stem cell transplantation. However, due to the toxicity associated to this treatment, improved therapies are recommended for DC patients. Here, we aimed at generating DC-like human hematopoietic stem cells in which the efficacy of innovative therapies could be investigated. Because X-linked DC is the most frequent form of the disease and is associated with an impaired expression of DKC1, we have generated DC-like hematopoietic stem cells based on the stable knock-down of DKC1 in human CD34+ cells with lentiviral vectors encoding for DKC1 short hairpin RNAs. At a molecular level, DKC1-interfered CD34+ cells showed a decreased expression of TERC, as well as a diminished telomerase activity and increased DNA damage, cell senescence, and apoptosis. Moreover, DKC1-interfered human CD34+ cells showed defective clonogenic ability and were incapable of repopulating the hematopoiesis of immunodeficient NSG mice. The development of DC-like hematopoietic stem cells will facilitate the understanding of the molecular and cellular basis of this inherited bone marrow failure syndrome and will serve as a platform to evaluate the efficacy of new hematopoietic therapies for DC.
Asunto(s)
Disqueratosis Congénita , Telomerasa , Animales , Proteínas de Ciclo Celular/genética , Disqueratosis Congénita/genética , Disqueratosis Congénita/terapia , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Mutación , Proteínas Nucleares/genética , Telomerasa/genética , Telomerasa/metabolismo , Telómero/metabolismoRESUMEN
Directed gene therapy mediated by nucleases has become a new alternative to lead targeted integration of therapeutic genes in specific regions in the genome. In this work, we have compared the efficiency of two nuclease types, TALEN and meganucleases (MN), to introduce an EGFP reporter gene in a specific site in a safe harbor locus on chromosome 21 in an intergenic region, named here SH6. The efficiency of targeted integration mediated by SH6v5-MN and SH6-TALEN in HEK-293H cells was up to 16.3 and 15.0%. A stable expression was observed both in the pool of transfected cells and in established pseudoclones, with no detection of off-target integrations by Southern blot. In human hematopoietic stem and progenitor CD34+ cells, the nucleofection process preserved the viability and clonogenic capacity of nucleofected cells, reaching up to 3.1% of specific integration of the transgene in colony forming cells when the SH6-TALEN was used, although no expression of the transgene could be found in these cells. Our results show the possibility to specifically integrate genes at the SH6 locus in CD34+ progenitor cells, although further improvements in the efficacy of the procedure are required before this approach could be used for the gene editing of hematopoietic stem cells in patients with hematopoietic diseases.
Asunto(s)
Terapia Genética , Células Madre Hematopoyéticas , Genes Reporteros , Humanos , Nucleasas de los Efectores Tipo Activadores de la Transcripción , TransgenesRESUMEN
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.
Asunto(s)
Anemia de Células Falciformes/terapia , Terapia Genética/tendencias , Enfermedades Hematológicas/terapia , Talasemia beta/terapia , Anemia de Células Falciformes/sangre , Células Sanguíneas/patología , Células Sanguíneas/trasplante , Vectores Genéticos/efectos adversos , Enfermedades Hematológicas/sangre , Enfermedades Hematológicas/patología , Trasplante de Células Madre Hematopoyéticas/tendencias , Células Madre Hematopoyéticas/citología , Humanos , Talasemia beta/sangreRESUMEN
Fanconi anemia (FA) is a DNA repair syndrome generated by mutations in any of the 22 FA genes discovered to date1,2. Mutations in FANCA account for more than 60% of FA cases worldwide3,4. 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 life5,6. 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.
Asunto(s)
Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Terapia Genética , Trasplante de Células Madre Hematopoyéticas , Adolescente , Adulto , Células de la Médula Ósea/citología , Niño , Preescolar , Anemia de Fanconi/genética , Anemia de Fanconi/fisiopatología , Femenino , Vectores Genéticos/genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Lactante , Lentivirus/genética , Masculino , Mutación/genética , España/epidemiología , Reparación del Gen Blanco , Transducción Genética , Adulto JovenRESUMEN
Ataxia telangiectasia (AT) is a genetic disease caused by mutations in the ATM gene but the mechanisms underlying AT are not completely understood. Key functions of the ATM protein are to sense and regulate cellular redox status and to transduce DNA double-strand break signals to downstream effectors. ATM-deficient cells show increased ROS accumulation, activation of p38 protein kinase, and increased levels of DNA damage. GSE24.2 peptide and a short derivative GSE4 peptide corresponding to an internal domain of Dyskerin have proved to induce telomerase activity, decrease oxidative stress, and protect from DNA damage in dyskeratosis congenita (DC) cells. We have found that expression of GSE24.2 and GSE4 in human AT fibroblast is able to decrease DNA damage, detected by γ-H2A.X and 53BP1 foci. However, GSE24.2/GSE4 expression does not improve double-strand break signaling and repair caused by the lack of ATM activity. In contrast, they cause a decrease in 8-oxoguanine and OGG1-derived lesions, particularly at telomeres and mitochondrial DNA, as well as in reactive oxygen species, in parallel with increased expression of SOD1. These cells also showed lower levels of IL6 and decreased p38 phosphorylation, decreased senescence and increased ability to divide for longer times. Additionally, these cells are more resistant to treatment with H202 and the radiomimetic-drug bleomycin. Finally, we found shorter telomere length (TL) in AT cells, lower levels of TERT expression, and telomerase activity that were also partially reverted by GSE4. These observations suggest that GSE4 may be considered as a new therapy for the treatment of AT that counteracts the cellular effects of high ROS levels generated in AT cells and in addition increases telomerase activity contributing to increased cell proliferation.
Asunto(s)
Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Fragmentos de Péptidos/metabolismo , Telómero/metabolismo , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/patología , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Línea Celular , Roturas del ADN de Doble Cadena , Daño del ADN , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Nanopartículas/química , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/química , Proteínas Nucleares/genética , Estrés Oxidativo/fisiología , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Telomerasa/metabolismo , Telómero/genética , Telómero/patologíaRESUMEN
The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34+ cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is â¼20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34+ cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34+ cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4-8 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol.
Asunto(s)
Elementos Transponibles de ADN , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Células Madre Hematopoyéticas/metabolismo , Animales , Supervivencia Celular , Citometría de Flujo , Expresión Génica , Humanos , Ratones , Ratones Noqueados , Retroviridae/genética , Transfección , TransgenesRESUMEN
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 FANCA-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.
Asunto(s)
Proteína del Grupo de Complementación C de la Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Terapia Genética/métodos , Vectores Genéticos , Trasplante de Células Madre Hematopoyéticas/métodos , Transducción Genética/métodos , Animales , Antígenos CD34/inmunología , Niño , Preescolar , Anemia de Fanconi/patología , Supervivencia de Injerto , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/patología , Xenoinjertos , Humanos , Lentivirus/genética , RatonesRESUMEN
NHEJ1-patients develop severe progressive lymphocytopenia and premature aging of hematopoietic stem cells (HSCs) at a young age. Here we show a patient with a homozygous-NHEJ1 mutation identified by whole exome-sequencing that developed severe pancytopenia and bone marrow aplasia correlating with the presence of short telomeres. The mutation resulted in a truncated protein. In an attempt to identify the mechanism behind the short telomere phenotype found in the NHEJ1-patient we downregulated NHEJ1 expression in 293T and CD34+cells. This downregulation resulted in reduced telomerase activity and decreased expression of several telomerase/shelterin genes. Interestingly, cell lines derived from two other NHEJ1-deficient patients with different mutations also showed increased p21 expression, inhibition in expression of several telomerase complex genes and shortened telomeres. Decrease in expression of telomerase/shelterin genes did not occur when we inhibited expression of other NHEJ genes mutated in SCID patients: DNA-PK, Artemis or LigaseIV. Because premature aging of HSCs is observed only in NHEJ1 patients, we propose that is the result of senescence induced by decreased expression of telomerase/shelterin genes that lead to an inhibition of telomerase activity. Previous reports failed to find this connection because of the use of patient´s cells immortalized by TERT expression or recombined telomeres by ALT pathway. In summary, defective regulation of telomere biology together with defective V(D)J recombination can negatively impact on the evolution of the disease in these patients. Identification of telomere shortening is important since it may open new therapeutic interventions for these patients by treatments aimed to recover the expression of telomerase genes.
Asunto(s)
Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Telomerasa/genética , Línea Celular , Niño , Enzimas Reparadoras del ADN/sangre , Proteínas de Unión al ADN/sangre , Regulación hacia Abajo , Expresión Génica , Humanos , Masculino , Mutación/genética , Telómero/genética , Telómero/metabolismo , Homeostasis del Telómero , Acortamiento del Telómero/genéticaRESUMEN
Pyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses.
Asunto(s)
Anemia Hemolítica Congénita no Esferocítica/genética , Anemia Hemolítica Congénita no Esferocítica/terapia , Células Eritroides/citología , Células Madre Pluripotentes Inducidas/metabolismo , Piruvato Quinasa/deficiencia , Piruvato Quinasa/genética , Errores Innatos del Metabolismo del Piruvato/genética , Errores Innatos del Metabolismo del Piruvato/terapia , Alelos , Secuencia de Bases , Recuento de Células , ADN Complementario/genética , Células Eritroides/metabolismo , Marcación de Gen , Terapia Genética , Humanos , Leucocitos Mononucleares/metabolismo , Recombinación GenéticaRESUMEN
Fanconi anemia is a DNA repair-deficiency syndrome mainly characterized by cancer predisposition and bone marrow failure. Trying to restore the hematopoietic function in these patients, lentiviral vector-mediated gene therapy trials have recently been proposed. However, because no insertional oncogenesis studies have been conducted so far in DNA repair-deficiency syndromes such as Fanconi anemia, we have carried out a genome-wide screening of lentiviral insertion sites after the gene correction of Fanca(-/-) hematopoietic stem cells (HSCs), using LAM-PCR and 454-pyrosequencing. Our studies first demonstrated that transduction of Fanca(-/-) HSCs with a lentiviral vector designed for clinical application efficiently corrects the phenotype of Fanconi anemia repopulating cells without any sign of toxicity. The identification of more than 6,500 insertion sites in primary and secondary recipients showed a polyclonal pattern of reconstitution, as well as a continuous turnover of corrected Fanca(-/-) HSC clones, without evidences of selection towards specific common integration sites. Taken together our data show, for the first time in a DNA repair-deficiency syndrome, that lentiviral vector-mediated gene therapy efficiently corrects the phenotype of affected HSCs and promotes a healthy pattern of clonal turnover in vivo. These studies will have a particular impact in the development of new gene therapy trials in patients affected by DNA repair syndromes, particularly in Fanconi anemia.
Asunto(s)
Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Terapia Genética/métodos , Vectores Genéticos/genética , Lentivirus/genética , Animales , Línea Celular , Reparación del ADN/genética , Anemia de Fanconi/genética , Células Madre Hematopoyéticas/citología , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Transducción Genética/métodosRESUMEN
Reprogramming technologies have emerged as a promising approach for future regenerative medicine. Here, we report on the establishment of a novel methodology allowing for the conversion of human fibroblasts into hematopoietic progenitor-like cells with macrophage differentiation potential. SOX2 overexpression in human fibroblasts, a gene found to be upregulated during hematopoietic reconstitution in mice, induced the rapid appearance of CD34+ cells with a concomitant upregulation of mesoderm-related markers. Profiling of cord blood hematopoietic progenitor cell populations identified miR-125b as a factor facilitating commitment of SOX2-generated CD34+ cells to immature hematopoietic-like progenitor cells with grafting potential. Further differentiation toward the monocytic lineage resulted in the appearance of CD14+ cells with functional phagocytic capacity. In vivo transplantation of SOX2/miR-125b-generated CD34+ cells facilitated the maturation of the engrafted cells toward CD45+ cells and ultimately the monocytic/macrophage lineage. Altogether, our results indicate that strategies combining lineage conversion and further lineage specification by in vivo or in vitro approaches could help to circumvent long-standing obstacles for the reprogramming of human cells into hematopoietic cells with clinical potential.
Asunto(s)
Diferenciación Celular/fisiología , Fibroblastos/citología , Monocitos/citología , Células Madre/citología , Animales , Antígenos CD34/metabolismo , Linaje de la Célula/fisiología , Células Cultivadas , Humanos , Antígenos Comunes de Leucocito/metabolismo , RatonesRESUMEN
Although there is an increasing interest in defining the role of DNA damage response mechanisms in cell reprogramming, the relevance of proteins participating in nonhomologous end joining (NHEJ), a major mechanism of DNA double-strand breaks repair, in this process remains to be investigated. Herein, we present data related to the reprogramming of primary mouse embryonic fibroblasts (MEF) from severe combined immunodeficient (Scid) mice defective in DNA-PKcs, a key protein for NHEJ. Reduced numbers of induced pluripotent stem cell (iPSC) colonies were generated from Scid cells using reprogramming lentiviral vectors (LV), being the reprogramming efficiency fourfold to sevenfold lower than that observed in wt cells. Moreover, these Scid iPSC-like clones were prematurely lost or differentiated spontaneously. While the Scid mutation neither reduce the proliferation rate nor the transduction efficacy of fibroblasts transduced with reprogramming LV, both the expression of SA-ß-Gal and of P16/INK(4a) senescence markers were highly increased in Scid versus wt MEFs during the reprogramming process, accounting for the reduced reprogramming efficacy of Scid MEFs. The use of improved Sleeping Beauty transposon/transposase systems allowed us, however, to isolate DNA-PKcs-deficient iPSCs which preserved their parental genotype and hypersensitivity to ionizing radiation. This new disease-specific iPSC model would be useful to understand the physiological consequences of the DNA-PKcs mutation during development and would help to improve current cell and gene therapy strategies for the disease.
Asunto(s)
Reprogramación Celular/genética , Reparación del ADN por Unión de Extremidades , Células Madre Pluripotentes Inducidas/fisiología , Animales , Ratones , Ratones Endogámicos BALB C , Ratones SCID , TransfecciónRESUMEN
The generation of patient-specific induced pluripotent stem cells (iPSCs) offers unprecedented opportunities for modeling and treating human disease. In combination with gene therapy, the iPSC technology can be used to generate disease-free progenitor cells of potential interest for autologous cell therapy. We explain a protocol for the reproducible generation of genetically corrected iPSCs starting from the skin biopsies of Fanconi anemia patients using retroviral transduction with OCT4, SOX2 and KLF4. Before reprogramming, the fibroblasts and/or keratinocytes of the patients are genetically corrected with lentiviruses expressing FANCA. The same approach may be used for other diseases susceptible to gene therapy correction. Genetically corrected, characterized lines of patient-specific iPSCs can be obtained in 4-5 months.
Asunto(s)
Técnicas de Transferencia de Gen , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Técnicas de Cultivo de Célula , Desdiferenciación Celular , Células Cultivadas , Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Terapia Genética/métodos , Humanos , Factor 4 Similar a Kruppel , Lentivirus/genética , Células Madre Pluripotentes/trasplante , Transducción Genética , Trasplante AutólogoRESUMEN
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.
Asunto(s)
Anemia de Fanconi/terapia , Terapia Genética , Vectores Genéticos/genética , Línea Celular , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Humanos , Mutación , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-vav , ARN Mensajero/genética , ARN Mensajero/metabolismo , Virus Formadores de Foco en el Bazo/genética , Virus Formadores de Foco en el Bazo/metabolismoRESUMEN
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.
Asunto(s)
Anemia de Fanconi/patología , Anemia de Fanconi/terapia , Células Madre Hematopoyéticas/citología , Células Madre Pluripotentes/citología , Línea Celular , Reprogramación Celular , Salud , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Pluripotentes/metabolismoRESUMEN
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).
Asunto(s)
Anemia de Fanconi/metabolismo , Anemia de Fanconi/terapia , Vectores Genéticos/genética , Células Madre Hematopoyéticas/metabolismo , Lentivirus/genética , Células Madre Mesenquimatosas/metabolismo , Antígenos CD34/metabolismo , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Línea Celular , Células Cultivadas , Anemia de Fanconi/patología , HumanosRESUMEN
The hematopoietic stem cells (HSCs) constitute an ideal target for the gene therapy of inherited diseases affecting the hematopoietic system. HSCs, however, constitute a very rare population of progenitor cells, most of which are out of cycle in normal bone marrow. To facilitate their transduction with gammaretro-viral or lentiviral vectors, HSCs are generally enriched using physical or pharmacologic methods. In this chapter we describe efficient procedures which are frequently used to enrich human and mouse HSCs, aiming at the transduction of these cells with adequate gene therapy vectors or the subsequent purification of particular HSCs by fluorescence-activated cell sorting.
Asunto(s)
Terapia Genética , Células Madre Hematopoyéticas/metabolismo , Separación Inmunomagnética , Animales , Antígenos CD34/inmunología , Fluorouracilo/administración & dosificación , Células Madre Hematopoyéticas/inmunología , Humanos , RatonesRESUMEN
Fanconi anemia (FA) is an inherited recessive DNA repair disorder mainly characterized by bone marrow failure and cancer predisposition. Studies in mosaic FA patients have shown that reversion of one inherited germ-line mutation resulting in a functional allele in one or a few hematopoietic stem cells (HSCs) can lead to the proliferation advantage of corrected cells, thus over time normalizing the hematologic status of the patient. In contrast to these observations, it is still unclear whether ex vivo genetic correction of FA HSCs also provides a similar proliferation advantage to FA HSCs. Using an FA mouse model with a marked hematopoietic phenotype, the FA-D1 (Brca2(Delta27/Delta27)) mice, we demonstrate that the lentivirus-mediated gene therapy of FA HSCs results in the progressive expansion of genetically corrected clones in mild-conditioned FA-D1 recipients. Consistent with these data, hematopoietic progenitors from FA recipients progressively became mitomycin C resistant and their chromosomal instability was reverted. No evidence of myelodysplasia, leukemias, or abnormal clonal repopulation was observed at multiple time points in primary or secondary recipients. Our results demonstrate that ectopic expression of BRCA2 confers a beneficial in vivo proliferation advantage to FA-D1 HSCs that enables the full hematopoietic repopulation of FA recipients with genetically corrected cells.
Asunto(s)
Proliferación Celular , Anemia de Fanconi/patología , Anemia de Fanconi/terapia , Terapia Genética/métodos , Células Madre Hematopoyéticas/citología , Animales , Proteína BRCA2/genética , Modelos Animales de Enfermedad , Mutación de Línea Germinal , RatonesRESUMEN
Recent studies of retroviral-mediated gene transfer have shown that retroviral integrations themselves may trigger nonmalignant clonal expansion of hematopoietic stem cells (HSCs) in transplant recipients. These observations suggested that previous conclusions of HSC dynamics based on gamma-retroviral gene marking should be confirmed with improved vectors having a more limited capacity to transactivate endogenous genes. Because of the low trans-activation activity of self-inactivating lentiviral vectors (LVs), we have investigated whether the LV marking of mouse HSCs induces a competitive repopulation advantage in recipients of serially transplants. As deduced from analyses conducted in primary and secondary recipients, we concluded that lentivirally transduced HSCs have no competitive repopulation advantages over untransduced HSCs. By linear amplification-mediated polymerase chain reaction (LAM-PCR) analysis, we characterized LV-targeted genes in HSC clones that engrafted up to quaternary recipients. Although 9 clones harbored integrations close to defined retroviral insertion sites, none was characterized as a common integration site, and none was present in HSC clones repopulating quaternary recipients. Taken together, our results show unaltered repopulation properties of HSCs transduced with LVs, and confirm early studies suggesting the natural capacity of a few HSC clones to generate a monoclonal or oligoclonal hematopoiesis in transplant recipients.