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1.
Cell Stem Cell ; 25(5): 639-653.e7, 2019 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-31631013

RESUMEN

Cellular stress responses serve as crucial decision points balancing persistence or culling of hematopoietic stem cells (HSCs) for lifelong blood production. Although strong stressors cull HSCs, the linkage between stress programs and self-renewal properties that underlie human HSC maintenance remains unknown, particularly at quiescence exit when HSCs must also dynamically shift metabolic state. Here, we demonstrate distinct wiring of the sphingolipidome across the human hematopoietic hierarchy and find that genetic or pharmacologic modulation of the sphingolipid enzyme DEGS1 regulates lineage differentiation. Inhibition of DEGS1 in hematopoietic stem and progenitor cells during the transition from quiescence to cellular activation with N-(4-hydroxyphenyl) retinamide activates coordinated stress pathways that coalesce on endoplasmic reticulum stress and autophagy programs to maintain immunophenotypic and functional HSCs. Thus, our work identifies a linkage between sphingolipid metabolism, proteostatic quality control systems, and HSC self-renewal and provides therapeutic targets for improving HSC-based cellular therapeutics.

2.
Blood ; 133(20): 2198-2211, 2019 05 16.
Artículo en Inglés | MEDLINE | ID: mdl-30796022

RESUMEN

There is a growing body of evidence that the molecular properties of leukemia stem cells (LSCs) are associated with clinical outcomes in acute myeloid leukemia (AML), and LSCs have been linked to therapy failure and relapse. Thus, a better understanding of the molecular mechanisms that contribute to the persistence and regenerative potential of LSCs is expected to result in the development of more effective therapies. We therefore interrogated functionally validated data sets of LSC-specific genes together with their known protein interactors and selected 64 candidates for a competitive in vivo gain-of-function screen to identify genes that enhanced stemness in human cord blood hematopoietic stem and progenitor cells. A consistent effect observed for the top hits was the ability to restrain early repopulation kinetics while preserving regenerative potential. Overexpression (OE) of the most promising candidate, the orphan gene C3orf54/INKA1, in a patient-derived AML model (8227) promoted the retention of LSCs in a primitive state manifested by relative expansion of CD34+ cells, accumulation of cells in G0, and reduced output of differentiated progeny. Despite delayed early repopulation, at later times, INKA1-OE resulted in the expansion of self-renewing LSCs. In contrast, INKA1 silencing in primary AML reduced regenerative potential. Mechanistically, our multidimensional confocal analysis found that INKA1 regulates G0 exit by interfering with nuclear localization of its target PAK4, with concomitant reduction of global H4K16ac levels. These data identify INKA1 as a novel regulator of LSC latency and reveal a link between the regulation of stem cell kinetics and pool size during regeneration.


Asunto(s)
Regulación Leucémica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Leucemia Mieloide Aguda/genética , Células Madre Neoplásicas/metabolismo , Animales , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Femenino , Humanos , Leucemia Mieloide Aguda/patología , Masculino , Ratones Endogámicos NOD , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/patología , Regulación hacia Arriba , Quinasas p21 Activadas/análisis
3.
Nat Genet ; 48(12): 1481-1489, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27776115

RESUMEN

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL). Here we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG is a hallmark of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt uses a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivation domains of ERG, but it inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia in which DUX4 deregulation results in loss of function of ERG, either by deletion or induced expression of an isoform that is a dominant-negative inhibitor of wild-type ERG function.


Asunto(s)
Transformación Celular Neoplásica/genética , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Reordenamiento Génico , Proteínas de Homeodominio/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Adulto , Transformación Celular Neoplásica/patología , Perfilación de la Expresión Génica , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Isoformas de Proteínas , Regulador Transcripcional ERG/genética , Adulto Joven
5.
PLoS One ; 11(4): e0153978, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27111551

RESUMEN

Orthopedic implant failure due to aseptic loosening and mechanical instability remains a major problem in total joint replacement. Improving osseointegration at the bone-implant interface may reduce micromotion and loosening. Bone sialoprotein (BSP) has been shown to enhance bone formation when coated onto titanium femoral implants and in rat calvarial defect models. However, the most appropriate method of BSP coating, the necessary level of BSP coating, and the effect of BSP coating on cell behavior remain largely unknown. In this study, BSP was covalently coupled to titanium surfaces via an aminosilane linker (APTES), and its properties were compared to BSP applied to titanium via physisorption and untreated titanium. Cell functions were examined using primary human osteoblasts (hOBs) and L929 mouse fibroblasts. Gene expression of specific bone turnover markers at the RNA level was detected at different intervals. Cell adhesion to titanium surfaces treated with BSP via physisorption was not significantly different from that of untreated titanium at any time point, whereas BSP application via covalent coupling caused reduced cell adhesion during the first few hours in culture. Cell migration was increased on titanium disks that were treated with higher concentrations of BSP solution, independent of the coating method. During the early phases of hOB proliferation, a suppressive effect of BSP was observed independent of its concentration, particularly when BSP was applied to the titanium surface via physisorption. Although alkaline phosphatase activity was reduced in the BSP-coated titanium groups after 4 days in culture, increased calcium deposition was observed after 21 days. In particular, the gene expression level of RUNX2 was upregulated by BSP. The increase in calcium deposition and the stimulation of cell differentiation induced by BSP highlight its potential as a surface modifier that could enhance the osseointegration of orthopedic implants. Both physisorption and covalent coupling of BSP are similarly effective, feasible methods, although a higher BSP concentration is recommended.


Asunto(s)
Sialoproteína de Unión a Integrina/química , Prótesis e Implantes , Titanio , Ortopedia , Propiedades de Superficie
6.
Cancer Cell ; 29(2): 214-28, 2016 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-26832662

RESUMEN

To investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance.


Asunto(s)
Células Madre Hematopoyéticas/patología , Leucemia Mieloide Aguda/patología , MicroARNs/fisiología , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Ratones SCID , MicroARNs/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Pronóstico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo
7.
Science ; 351(6269): aab2116, 2016 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-26541609

RESUMEN

In a classical view of hematopoiesis, the various blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. We developed a cell-sorting scheme to resolve myeloid (My), erythroid (Er), and megakaryocytic (Mk) fates from single CD34(+) cells and then mapped the progenitor hierarchy across human development. Fetal liver contained large numbers of distinct oligopotent progenitors with intermingled My, Er, and Mk fates. However, few oligopotent progenitor intermediates were present in the adult bone marrow. Instead, only two progenitor classes predominate, multipotent and unipotent, with Er-Mk lineages emerging from multipotent cells. The developmental shift to an adult "two-tier" hierarchy challenges current dogma and provides a revised framework to understand normal and disease states of human hematopoiesis.


Asunto(s)
Linaje de la Célula/fisiología , Células Eritroides/citología , Hematopoyesis/fisiología , Células Progenitoras de Megacariocitos/citología , Megacariocitos/citología , Células Mieloides/citología , Adulto , Antígenos CD34/análisis , Linaje de la Célula/genética , Separación Celular , Células Cultivadas , Sangre Fetal/citología , Perfilación de la Expresión Génica , Hematopoyesis/genética , Humanos , Hígado/citología , Hígado/embriología , Células Madre Multipotentes/citología , Transcripción Genética
8.
Stem Cells Dev ; 24(6): 714-23, 2015 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-25517513

RESUMEN

Hematopoietic stem cells (HSCs) are an important target cell population for gene therapy since they can reconstitute the entire hematopoietic system. HSC-enriched cell populations can be recognized based on cell surface marker expression, such as CD34, which is broadly expressed on immature and partially differentiated cells. In mice, co-expression of CD34 and CD105 was previously shown to be relatively more specific for the most immature, long-term repopulating HSCs. Here, we evaluated whether CD105, which is expressed on 30%-80% of CD34(+) cells, is a marker also for human long-term repopulating HSCs. Therefore, we tracked the mature progeny of CD34(+) cells transduced with the CD105-targeted lentiviral vector CD105-LV in xenotolerant mice. Transduction was blocked with soluble CD105 protein confirming specificity. Importantly, CD105-LV transduced human CD34(+) cells engrafted in NOD-scid IL2Rγ(-/-) mice with up to 20% reporter gene-positive cells detected long term in all human hematopoietic lineages in bone marrow (BM), spleen, and blood. In addition, competitive repopulation experiments in mice showed a superior engraftment of CD105-LV transduced CD34(+) cells in BM and spleen compared with cells transduced with a conventional nontargeted lentiviral vector. Thus, human CD34(+)/CD105(+) cells are enriched for early HSCs with high repopulating capacity. Targeting this cell population with CD105-LV offers a novel gene transfer strategy to reach high engraftment rates of transduced cells and highlights the applicability of receptor-targeted vectors to trace cell subsets offering an alternative to prospective isolation by surface markers.


Asunto(s)
Antígenos CD/metabolismo , Células Madre Hematopoyéticas/metabolismo , Receptores de Superficie Celular/metabolismo , Trasplante de Células Madre/métodos , Animales , Antígenos CD/genética , Antígenos CD34/genética , Antígenos CD34/metabolismo , Diferenciación Celular , Endoglina , Marcación de Gen , Células Madre Hematopoyéticas/citología , Humanos , Subunidad gamma Común de Receptores de Interleucina/genética , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptores de Superficie Celular/genética
9.
Mol Ther ; 23(1): 63-70, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25189742

RESUMEN

Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cells (HSCs) as entry receptor, transfers genes preferentially into cells with high engraftment capability. Transduction of unstimulated CD34(+) cells with CD133-LV resulted in gene marking of cells with competitive proliferative advantage in vitro and in immunodeficient mice. The CD133-LV-transduced population contained significantly more cells with repopulating capacity than cells transduced with vesicular stomatitis virus (VSV)-LV, a lentiviral vector pseudotyped with the vesicular stomatitis virus G protein. Upon transfer of a barcode library, CD133-LV-transduced cells sustained gene marking in vivo for a prolonged period of time with a 6.7-fold higher recovery of barcodes compared to transduced control cells. Moreover, CD133-LV-transduced cells were capable of repopulating secondary recipients. Lastly, we show that this targeting strategy can be used for transfer of a therapeutic gene into CD34(+) cells obtained from patients suffering of X-linked chronic granulomatous disease. In conclusion, direct gene transfer into CD133(+) cells allows for sustained long-term engraftment of gene corrected cells.


Asunto(s)
Antígenos CD/genética , Terapia Genética/métodos , Glicoproteínas/genética , Células Madre Hematopoyéticas/inmunología , Lentivirus/genética , Péptidos/genética , Antígeno AC133 , Animales , Antígenos CD/inmunología , Antígenos CD34/genética , Antígenos CD34/inmunología , Expresión Génica , Vectores Genéticos , Glicoproteínas/inmunología , Enfermedad Granulomatosa Crónica/genética , Enfermedad Granulomatosa Crónica/inmunología , Enfermedad Granulomatosa Crónica/patología , Enfermedad Granulomatosa Crónica/terapia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Péptidos/inmunología , Cultivo Primario de Células , Transducción Genética , Virus de la Estomatitis Vesicular Indiana/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo
10.
Curr Gene Ther ; 14(6): 447-60, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25245086

RESUMEN

Several Phase I/II clinical trials aiming at the correction of X-linked CGD by gene transfer into hematopoietic stem cells (HSCs) have demonstrated the therapeutic potential of gene modified autologous HSCs for the treatment of CGD. Resolution of therapy-resistant bacterial and fungal infections in liver, lung and spinal canal of CGD patients were clearly documented in all trials. However, clinical benefits were not sustained over time due to the failure of gene transduced cells to engraft long-term. Moreover, severe adverse effects were observed in some of the treated patients due to insertional mutagenesis leading to the activation of growth promoting genes and to myeloid malignancy. These setbacks fostered the development of novel safety and efficacy improved vectors that have already entered or are about to enter the clinics. Meanwhile, ongoing research is constantly refining the CGD disease phenotype, including the definition of factors that may explain the unique engraftment phenotype observed in CGD gene therapy trials. This review provides a condensed overview on the current knowledge of the molecular pathomechanisms and clinical manifestations of CGD and summarizes the lessons learned from clinical gene therapy trials, the preclinical progress in vector design and the future perspectives for the gene therapy of CGD.


Asunto(s)
Terapia Genética , Vectores Genéticos/uso terapéutico , Enfermedad Granulomatosa Crónica/genética , Enfermedad Granulomatosa Crónica/terapia , Animales , Humanos
11.
Antioxid Redox Signal ; 21(11): 1605-19, 2014 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-24762207

RESUMEN

SIGNIFICANCE: Blood forming, hematopoietic stem cells (HSCs) mostly reside in the bone marrow in a quiescent, nonmotile state via adhesion interactions with stromal cells and macrophages. Quiescent, proliferating, and differentiating stem cells have different metabolism, and accordingly different amounts of intracellular reactive oxygen species (ROS). Importantly, ROS is not just a byproduct of metabolism, but also plays a role in stem cell state and function. RECENT ADVANCES: ROS levels are dynamic and reversibly dictate enhanced cycling and myeloid bias in ROS(high) short-term repopulating stem cells, and ROS(low) quiescent long-term repopulating stem cells. Low levels of ROS, regulated by intrinsic factors such as cell respiration or nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) activity, or extrinsic factors such as stem cell factor or prostaglandin E2 are required for maintaining stem cell self-renewal. High ROS levels, due to stress and inflammation, induce stem cell differentiation and enhanced motility. CRITICAL ISSUES: Stem cells need to be protected from high ROS levels to avoid stem cell exhaustion, insufficient host immunity, and leukemic transformation that may occur during chronic inflammation. However, continuous low ROS production will lead to lack of stem cell function and opportunistic infections. Ultimately, balanced ROS levels are crucial for maintaining the small stem cell pool and host immunity, both in homeostasis and during stress situations. FUTURE DIRECTIONS: Deciphering the signaling pathway of ROS in HSC will provide a better understanding of ROS roles in switching HSC from quiescence to activation and vice versa, and will also shed light on the possible roles of ROS in leukemia initiation and development.


Asunto(s)
Células de la Médula Ósea , Médula Ósea/metabolismo , Diferenciación Celular , Movimiento Celular , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Nicho de Células Madre/fisiología , Animales , Ciclo Celular , Proliferación Celular , Neoplasias Hematológicas/metabolismo , Humanos , Inflamación/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo
12.
Mol Ther Methods Clin Dev ; 1: 14037, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-26015977

RESUMEN

Innovative approaches for the treatment of rare inherited diseases are hampered by limited availability of patient derived samples for preclinical research. This also applies for the evaluation of novel vector systems for the gene therapy of monogenic hematological diseases like X-linked chronic granulomatous disease (X-CGD), a severe primary immunodeficiency caused by mutations in the gp91(phox) subunit of the phagocytic NADPH oxidase. Since current gene therapy protocols involve ex vivo gene modification of autologous CD34(+) hematopoietic stem cells (HSC), the ideal preclinical model should simulate faithfully this procedure. However, the low availability of patient-derived CD34(+) cells limits the feasibility of this approach. Here, we describe a straightforward experimental strategy that circumvents this limitation. The knock down of gp91(phox) expression upon lentiviral delivery of shRNAs into CD34(+) cells from healthy donors generates sufficient amounts of X-CGD CD34(+) cells which subsequently can be used for the evaluation of novel gene therapeutic strategies using a codon-optimized gp91(phox) transgene. We have used this strategy to test the potential of a novel gene therapy vector for X-CGD.

13.
EMBO Mol Med ; 5(11): 1642-61, 2013 11.
Artículo en Inglés | MEDLINE | ID: mdl-24106209

RESUMEN

The first gene therapy clinical trials were initiated more than two decades ago. In the early days, gene therapy shared the fate of many experimental medicine approaches and was impeded by the occurrence of severe side effects in a few treated patients. The understanding of the molecular and cellular mechanisms leading to treatment- and/or vector-associated setbacks has resulted in the development of highly sophisticated gene transfer tools with improved safety and therapeutic efficacy. Employing these advanced tools, a series of Phase I/II trials were started in the past few years with excellent clinical results and no side effects reported so far. Moreover, highly efficient gene targeting strategies and site-directed gene editing technologies have been developed and applied clinically. With more than 1900 clinical trials to date, gene therapy has moved from a vision to clinical reality. This review focuses on the application of gene therapy for the correction of inherited diseases, the limitations and drawbacks encountered in some of the early clinical trials and the revival of gene therapy as a powerful treatment option for the correction of monogenic disorders.


Asunto(s)
Terapia Genética , Ensayos Clínicos como Asunto , Técnicas de Transferencia de Gen/tendencias , Terapia Genética/métodos , Terapia Genética/tendencias , Humanos
14.
Hum Gene Ther Clin Dev ; 24(2): 86-98, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23845071

RESUMEN

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by impaired antimicrobial activity in phagocytic cells. As a monogenic disease affecting the hematopoietic system, CGD is amenable to gene therapy. Indeed in a phase I/II clinical trial, we demonstrated a transient resolution of bacterial and fungal infections. However, the therapeutic benefit was compromised by the occurrence of clonal dominance and malignant transformation demanding alternative vectors with equal efficacy but safety-improved features. In this work we have developed and tested a self-inactivating (SIN) gammaretroviral vector (SINfes.gp91s) containing a codon-optimized transgene (gp91(phox)) under the transcriptional control of a myeloid promoter for the gene therapy of the X-linked form of CGD (X-CGD). Gene-corrected cells protected X-CGD mice from Aspergillus fumigatus challenge at low vector copy numbers. Moreover, the SINfes.gp91s vector generates substantial amounts of superoxide in human cells transplanted into immunodeficient mice. In vitro genotoxicity assays and longitudinal high-throughput integration site analysis in transplanted mice comprising primary and secondary animals for 11 months revealed a safe integration site profile with no signs of clonal dominance.


Asunto(s)
Gammaretrovirus/genética , Vectores Genéticos/metabolismo , Enfermedad Granulomatosa Crónica/terapia , Animales , Aspergillus fumigatus/patogenicidad , Células Cultivadas , Metilación de ADN , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Terapia Genética , Vectores Genéticos/genética , Humanos , Enfermedades Pulmonares/microbiología , Enfermedades Pulmonares/patología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , NADPH Oxidasa 2 , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Fenotipo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-fes/genética , Superóxidos/metabolismo
15.
Curr Opin Hematol ; 20(3): 237-44, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23567340

RESUMEN

PURPOSE OF REVIEW: Fibroblast growth factor (FGF) signaling activates many bone marrow cell types, including various stem cells, osteoblasts, and osteoclasts. However, the role of FGF signaling in regulation of normal and leukemic stem cells is poorly understood. This review highlights the physiological roles of FGF signaling in regulating bone marrow mesenchymal and hematopoietic stem and progenitor cells (MSPCs and HSPCs) and their dynamic microenvironment. In addition, this review summarizes the recent studies which provide an overview of FGF-activated mechanisms regulating physiological stem cell maintenance, self-renewal, and motility. RECENT FINDINGS: Current results indicate that partial deficiencies in FGF signaling lead to mild defects in hematopoiesis and bone remodeling. However, FGF signaling was shown to be crucial for stem cell self-renewal and for proper hematopoietic poststress recovery. FGF signaling activation was shown to be important also for rapid AMD3100 or post 5-fluorouracil-induced HSPC mobilization. In vivo, FGF-2 administration successfully expanded both MSPCs and HSPCs. FGF-induced expansion was characterized by enhanced HSPC cycling without further exhaustion of the stem cell pool. In addition, FGF signaling expands and remodels the supportive MSPC niche cells. Finally, FGF signaling is constitutively activated in many leukemias, suggesting that malignant HSPCs exploit this pathway for their constant expansion and for remodeling a malignant-supportive microenvironment. SUMMARY: The summarized studies, concerning regulation of stem cells and their microenvironment, suggest that FGF signaling manipulation can serve to improve current clinical stem cell mobilization and transplantation protocols. In addition, it may help to develop therapies specifically targeting leukemic stem cells and their supportive microenvironment.


Asunto(s)
Remodelación Ósea/fisiología , Factores de Crecimiento de Fibroblastos/fisiología , Células Madre Hematopoyéticas/fisiología , Células Madre Mesenquimatosas/fisiología , Transducción de Señal/fisiología , Células Madre Hematopoyéticas/citología , Humanos , Células Madre Mesenquimatosas/citología , Microambiente Tumoral/fisiología
16.
Mol Ther ; 21(3): 648-61, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23207695

RESUMEN

Comparative integrome analysis has revealed that the most neutral integration pattern among retroviruses is attributed to alpharetroviruses. We chose X-linked chronic granulomatous disease (X-CGD) as model to evaluate the potential of self-inactivating (SIN) alpharetroviral vectors for gene therapy of monogenic diseases. Therefore, we combined the alpharetroviral vector backbone with the elongation factor-1α short promoter, both considered to possess a low genotoxic profile, to drive transgene (gp91(phox)) expression. Following efficient transduction transgene expression was sustained and provided functional correction of the CGD phenotype in a cell line model at low vector copy number. Further analysis in a murine X-CGD transplantation model revealed gene-marking of bone marrow cells and oxidase positive granulocytes in peripheral blood. Transduction of human X-CGD CD34+ cells provided functional correction up to wild-type levels and long-term expression upon transplantation into a humanized mouse model. In contrast to lentiviral vectors, no aberrantly spliced transcripts containing cellular exons fused to alpharetroviral sequences were found in transduced cells, implying that the safety profile of alpharetroviral vectors may extend beyond their neutral integration profile. Taken together, this highlights the potential of this SIN alpharetroviral system as a platform for new candidate vectors for future gene therapy of hematopoietic disorders.


Asunto(s)
Alpharetrovirus/genética , Terapia Genética/métodos , Vectores Genéticos , Enfermedad Granulomatosa Crónica/terapia , Empalme del ARN , Animales , Células de la Médula Ósea , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN , Modelos Animales de Enfermedad , Granulocitos , Enfermedad Granulomatosa Crónica/genética , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción Genética , Transgenes
17.
Mol Ther ; 20(5): 1022-32, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22334016

RESUMEN

Comparative integrome analyses have highlighted alpharetroviral vectors with a relatively neutral, and thus favorable, integration spectrum. However, previous studies used alpharetroviral vectors harboring viral coding sequences and intact long-terminal repeats (LTRs). We recently developed self-inactivating (SIN) alpharetroviral vectors with an advanced split-packaging design. In a murine bone marrow (BM) transplantation model we now compared alpharetroviral, gammaretroviral, and lentiviral SIN vectors and showed that all vectors transduced hematopoietic stem cells (HSCs), leading to comparable, sustained multilineage transgene expression in primary and secondary transplanted mice. Alpharetroviral integrations were decreased near transcription start sites, CpG islands, and potential cancer genes compared with gammaretroviral, and decreased in genes compared with lentiviral integrations. Analyzing the transcriptome and intragenic integrations in engrafting cells, we observed stronger correlations between in-gene integration targeting and transcriptional activity for gammaretroviral and lentiviral vectors than for alpharetroviral vectors. Importantly, the relatively "extragenic" alpharetroviral integration pattern still supported long-term transgene expression upon serial transplantation. Furthermore, sensitive genotoxicity studies revealed a decreased immortalization incidence compared with gammaretroviral and lentiviral SIN vectors. We conclude that alpharetroviral SIN vectors have a favorable integration pattern which lowers the risk of insertional mutagenesis while supporting long-term transgene expression in the progeny of transplanted HSCs.


Asunto(s)
Alpharetrovirus/genética , Vectores Genéticos , Células Madre Hematopoyéticas/metabolismo , Transgenes , Animales , Línea Celular , Islas de CpG , Gammaretrovirus/genética , Marcación de Gen , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Humanos , Lentivirus/genética , Ratones , Mutagénesis Insercional , Factores de Riesgo , Secuencias Repetidas Terminales , Sitio de Iniciación de la Transcripción , Transcriptoma/genética , Transducción Genética
18.
Virus Res ; 151(2): 220-8, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20621673

RESUMEN

The adenovirus major late promoter (MLP) generates a primary transcript that undergoes a complex pattern of regulated alternative RNA splicing and polyadenylation events. The late-specific activation of the MLP requires binding of two infected-cell specific transcription factor complexes, DEF-A and DEF-B, to the so-called DE sequence located downstream of the MLP start site. Previous studies have shown that DEF-B is a homodimer of the viral IVa2 protein and suggested that DEF-A is a heterodimer of IVa2 and an unknown protein. Two proteins from the adenoviral L4 unit have been suggested as DEF-A candidates. Here we have examined L4-22K and L4-33K for possible DEF-A activity. We show that L4-22K stimulates transcription from the MLP in a DE sequence dependent manner both in vivo and in vitro, and that L4-22K binds to the DE sequence in vitro. Further, the position of the L4-22K DNA binding site in a promoter does not appear to be critical for function. Thus, tethering L4-22K either to a position upstream or downstream of the MLP start site, or upstream of a minimal E1B promoter, resulted in an activation of transcription. We also show that the viral pIX promoter is a natural target, activated by L4-22K. Collectively, our results are compatible with the hypothesis that L4-22K may be the elusive component of DEF-A that partakes in activation of the MLP.


Asunto(s)
Adenoviridae/fisiología , ADN Viral/metabolismo , Regulación Viral de la Expresión Génica , Regiones Promotoras Genéticas , Transcripción Genética , Proteínas no Estructurales Virales/metabolismo , Sitios de Unión , Línea Celular , Humanos , Unión Proteica
19.
J Virol ; 84(8): 4083-8, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20147393

RESUMEN

Cytomegalovirus (CMV) infection in patients receiving hematopoietic stem cell transplants (HSCT) is associated with morbidity and mortality. Adoptive T cell immunotherapy has been used to treat viral reactivation but is hardly feasible in high-risk constellations of CMV-positive HSCT patients and CMV-negative stem cell donors. We endowed human effector T cells with a chimeric immunoreceptor (cIR) directed against CMV glycoprotein B. These cIR-engineered primary T cells mediated antiviral effector functions such as cytokine production and cytolysis. This first description of cIR-redirected CMV-specific T cells opens up a new perspective for HLA-independent immunotherapy of CMV infection in high-risk patients.


Asunto(s)
Citomegalovirus/inmunología , Citomegalovirus/fisiología , Receptores Inmunológicos/genética , Linfocitos T/inmunología , Linfocitos T/virología , Células Cultivadas , Infecciones por Citomegalovirus/terapia , Humanos , Inmunoterapia/métodos , Receptores Inmunológicos/metabolismo
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