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1.
Blood ; 144(7): 729-741, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-38805639

RESUMO

ABSTRACT: Loss of long-term hematopoietic stem cell (LT-HSC) function ex vivo hampers the success of clinical protocols that rely on culture. However, the kinetics and mechanisms through which this occurs remain incompletely characterized. In this study, through time-resolved single-cell RNA sequencing, matched in vivo functional analysis, and the use of a reversible in vitro system of early G1 arrest, we defined the sequence of transcriptional and functional events that occur during the first ex vivo division of human LT-HSCs. We demonstrated that the sharpest loss in LT-HSC repopulation capacity happens early on, between 6 and 24 hours of culture, before LT-HSCs commit to cell cycle progression. During this time window, LT-HSCs adapt to the culture environment, limit the global variability in gene expression, and transiently upregulate gene networks involved in signaling and stress responses. From 24 hours, LT-HSC progression past early G1 contributes to the establishment of differentiation programs in culture. However, contrary to the current assumptions, we demonstrated that the loss of HSC function ex vivo is independent of cell cycle progression. Finally, we showed that targeting LT-HSC adaptation to culture by inhibiting the early activation of JAK/STAT signaling improves HSC long-term repopulating function ex vivo. Collectively, our study demonstrated that controlling early LT-HSC adaptation to ex vivo culture, for example, via JAK inhibition, is critically important to improve HSC gene therapy and expansion protocols.


Assuntos
Ciclo Celular , Células-Tronco Hematopoéticas , Humanos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células Cultivadas , Transdução de Sinais , Diferenciação Celular , Técnicas de Cultura de Células/métodos , Adaptação Fisiológica
2.
Int J Mol Sci ; 20(18)2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31491876

RESUMO

Fabry disease (FD) is caused by mutations in the GLA gene that encodes lysosomal α-galactosidase-A (α-gal-A). A number of pathogenic mechanisms have been proposed and these include loss of mitochondrial respiratory chain activity. For FD, gene therapy is beginning to be applied as a treatment. In view of the loss of mitochondrial function reported in FD, we have considered here the impact of loss of mitochondrial respiratory chain activity on the ability of a GLA lentiviral vector to increase cellular α-gal-A activity and participate in cross correction. Jurkat cells were used in this study and were exposed to increasing viral copies. Intracellular and extracellular enzyme activities were then determined; this in the presence or absence of the mitochondrial complex I inhibitor, rotenone. The ability of cells to take up released enzyme was also evaluated. Increasing transgene copies was associated with increasing intracellular α-gal-A activity but this was associated with an increase in Km. Release of enzyme and cellular uptake was also demonstrated. However, in the presence of rotenone, enzyme release was inhibited by 37%. Excessive enzyme generation may result in a protein with inferior kinetic properties and a background of compromised mitochondrial function may impair the cross correction process.


Assuntos
Complexo I de Transporte de Elétrons/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , alfa-Galactosidase/biossíntese , Linhagem Celular , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/metabolismo , Doença de Fabry/genética , Doença de Fabry/metabolismo , Dosagem de Genes , Expressão Gênica , Humanos , Células Jurkat , Lisossomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Transdução Genética , Transgenes , alfa-Galactosidase/genética
3.
Mol Ther ; 27(10): 1706-1717, 2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31526597

RESUMO

The field of cell and gene therapy (GT) is expanding rapidly and there is undoubtedly a wave of enthusiasm and anticipation for what these treatments could achieve next. Here we assessed the worldwide landscape of GT assets currently in early clinical development (clinical trial phase 1/2 or about to enter clinical trial). We included all gene therapies, i.e., strategies that modify an individual's protein make-up by introducing exogenous nucleic acid or nucleic acid modifiers, regardless of delivery. Unmodified cell therapies, oncology therapies (reviewed elsewhere), and vaccine programs (distinct therapeutic strategy) were not included. Using a December 31, 2018 cutoff date, we identified 336 gene therapies being developed for 138 different indications covering 165 genetic targets. In all, we found that the early clinical GT landscape comprises a very disparate group of drug candidates in terms of indications, organizations, and delivery methods. We also highlight interesting trends, revealing the evolution of the field toward in vivo therapies and adeno-associated virus vector-based delivery systems. It will be interesting to witness what proportion of this current list effectively translates into new medicines.


Assuntos
Sistemas de Liberação de Medicamentos/classificação , Terapia Genética/métodos , Ensaios Clínicos como Assunto , Vetores Genéticos/administração & dosagem , Humanos , Terapia de Alvo Molecular
4.
Mol Ther Nucleic Acids ; 12: 626-634, 2018 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-30081233

RESUMO

Viral vectors are rapidly being developed for a range of applications in research and gene therapy. Prototype foamy virus (PFV) vectors have been described for gene therapy, although their use has mainly been restricted to ex vivo stem cell modification. Here we report direct in vivo transgene delivery with PFV vectors carrying reporter gene constructs. In our investigations, systemic PFV vector delivery to neonatal mice gave transgene expression in the heart, xiphisternum, liver, pancreas, and gut, whereas intracranial administration produced brain expression until animals were euthanized 49 days post-transduction. Immunostaining and confocal microscopy analysis of injected brains showed that transgene expression was highly localized to hippocampal architecture despite vector delivery being administered to the lateral ventricle. This was compared with intracranial biodistribution of lentiviral vectors and adeno-associated virus vectors, which gave a broad, non-specific spread through the neonatal mouse brain without regional localization, even when administered at lower copy numbers. Our work demonstrates that PFV can be used for neonatal gene delivery with an intracranial expression profile that localizes to hippocampal neurons, potentially because of the mitotic status of the targeted cells, which could be of use for research applications and gene therapy of neurological disorders.

5.
Exp Hematol ; 57: 21-29, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28911908

RESUMO

Integration-deficient lentiviruses (IdLVs) deliver genes effectively to tissues but are lost rapidly from dividing cells. This property can be harnessed to express transgenes transiently to manipulate cell biology. Here, we demonstrate the utility of short-term gene expression to improve functional potency of hematopoietic stem and progenitor cells (HSPCs) during transplantation by delivering HOXB4 and Angptl3 using IdLVs to enhance the engraftment of HSPCs. Constitutive overexpression of either of these genes is likely to be undesirable, but the transient nature of IdLVs reduces this risk and those associated with unsolicited gene expression in daughter cells. Transient expression led to increased multilineage hematopoietic engraftment in in vivo competitive repopulation assays without the side effects reported in constitutive overexpression models. Adult stem cell fate has not been programmed previously using IdLVs, but we demonstrate that these transient gene expression tools can produce clinically relevant alterations or be applied to investigate basic biology.


Assuntos
Vetores Genéticos/genética , Células-Tronco Hematopoéticas/fisiologia , Lentivirus/genética , Transdução Genética , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina/biossíntese , Proteínas Semelhantes a Angiopoietina/genética , Animais , Linhagem da Célula , Regulação da Expressão Gênica , Genes Reporter , Sobrevivência de Enxerto , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Células K562 , Camundongos , Camundongos Endogâmicos C57BL , Quimera por Radiação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transgenes
6.
Sci Rep ; 7(1): 12475, 2017 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-28963568

RESUMO

The generation of T cells from pluripotent stem cells (PSCs) is attractive for investigating T cell development and validating genome editing strategies in vitro. X-linked severe combined immunodeficiency (X-SCID) is an immune disorder caused by mutations in the IL2RG gene and characterised by the absence of T and NK cells in patients. IL2RG encodes the common gamma chain, which is part of several interleukin receptors, including IL-2 and IL-7 receptors. To model X-SCID in vitro, we generated a mouse embryonic stem cell (ESC) line in which a disease-causing human IL2RG gene variant replaces the endogenous Il2rg locus. We developed a stage-specific T cell differentiation protocol to validate genetic correction of the common G691A mutation with transcription activator-like effector nucleases. While all ESC clones could be differentiated to hematopoietic precursor cells, stage-specific analysis of T cell maturation confirmed early arrest of T cell differentiation at the T cell progenitor stage in X-SCID cells. In contrast, genetically corrected ESCs differentiated to CD4 + or CD8 + single-positive T cells, confirming correction of the cellular X-SCID phenotype. This study emphasises the value of PSCs for disease modelling and underlines the significance of in vitro models as tools to validate genome editing strategies before clinical application.


Assuntos
Edição de Genes/métodos , Células-Tronco Hematopoéticas/imunologia , Subunidade gama Comum de Receptores de Interleucina/genética , Células-Tronco Embrionárias Murinas/imunologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Substituição de Aminoácidos , Animais , Diferenciação Celular , Modelos Animais de Doenças , Expressão Gênica , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Interleucina-2/farmacologia , Interleucina-7/genética , Interleucina-7/imunologia , Interleucina-7/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Camundongos , Camundongos SCID , Camundongos Transgênicos , Terapia de Alvo Molecular , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/patologia , Mutação , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologia , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/imunologia , Transgenes , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/patologia
7.
Sci Rep ; 7: 44775, 2017 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-28303972

RESUMO

Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a 'template-switching' lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD.


Assuntos
Distrofina/genética , Distrofina/uso terapêutico , Terapia Genética , Vetores Genéticos/metabolismo , Lentivirus/genética , Linhagem Celular , Pré-Escolar , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Mioblastos/metabolismo , Mioblastos/patologia , Moldes Genéticos , Transdução Genética , Transgenes
8.
Sci Rep ; 7(1): 79, 2017 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-28250438

RESUMO

Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a 'template-switching' lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD.


Assuntos
Distrofina/genética , Distrofina/metabolismo , Lentivirus/genética , Distrofia Muscular de Duchenne/genética , Mioblastos Esqueléticos/metabolismo , Células Cultivadas , Empacotamento do DNA , Terapia Genética , Vetores Genéticos/genética , Células HEK293 , Células HeLa , Humanos , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Mioblastos Esqueléticos/patologia , Recombinação Genética , Transdução Genética
9.
J Hepatol ; 66(5): 1001-1011, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28082148

RESUMO

BACKGROUND & AIMS: In the normal liver, hepatocytes form a uniquely polarised cell layer that enables movement of solutes from sinusoidal blood to canalicular bile. Whilst several cholestatic liver diseases with defects of hepatocyte polarity have been identified, the molecular mechanisms of pathogenesis are not well defined. One example is arthrogryposis, renal dysfunction and cholestasis syndrome, which in most patients is caused by VPS33B mutations. VPS33B is a protein involved in membrane trafficking that interacts with RAB11A at recycling endosomes. To understand the pathways that regulate hepatocyte polarity better, we investigated VPS33B deficiency using a novel mouse model with a liver-specific Vps33b deletion. METHODS: To assess functional polarity, plasma and bile samples were collected from Vps33b liver knockout (Vps33bfl/fl-AlfpCre) and control (Vps33bfl/fl) mice; bile components or injected substrates were quantitated by mass spectrometry or fluorometry. For structural analysis, livers underwent light and transmission electron microscopy. Apical membrane and tight junction protein localisation was assessed by immunostaining. Adeno-associated virus vectors were used for in vivo gene rescue experiments. RESULTS: Like patients, Vps33bfl/fl-AlfpCre mice showed mislocalisation of ATP-binding cassette proteins that are specifically trafficked to the apical membrane via Rab11a-positive recycling endosomes. This was associated with retention of bile components in blood. Loss of functional tight junction integrity and depletion of apical microvilli were seen in knockout animals. Gene transfer partially rescued these defects. CONCLUSIONS: Vps33b has a key role in establishing structural and functional aspects of hepatocyte polarity and may be a target for gene replacement therapy. LAY SUMMARY: Hepatocytes are liver cells with tops and bottoms; that is, they are polarised. At their bottoms they absorb substances from blood. They then, at their tops, secrete these substances and their metabolites into bile. When polarity is lost, this directional flow of substances from blood to bile is disrupted and liver disease follows. In this study, using a new mouse model with a liver-specific mutation of Vps33b, the mouse version of a gene that is mutated in most patients with arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome, we investigated how the Vps33b gene product contributes to establishing hepatocyte polarity. We identified in these mice abnormalities similar to those in children with ARC syndrome. Gene transfer could partly reverse the mouse abnormalities. Our work contributes to the understanding of VPS33B disease and hepatocyte polarity in general, and may point towards gene transfer mediated treatment of ARC liver disease.


Assuntos
Polaridade Celular , Hepatócitos/fisiologia , Proteínas de Transporte Vesicular/fisiologia , Animais , Artrogripose/patologia , Artrogripose/terapia , Ácidos e Sais Biliares/sangue , Colestase/patologia , Colestase/terapia , Colesterol/sangue , Terapia Genética , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Insuficiência Renal/patologia , Insuficiência Renal/terapia , Junções Íntimas/fisiologia , Proteínas de Transporte Vesicular/genética
10.
Am J Physiol Lung Cell Mol Physiol ; 312(2): L258-L267, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-27979861

RESUMO

Air-liquid interface (ALI) culture of primary airway epithelial cells enables mucociliary differentiation providing an in vitro model of the human airway, but their proliferative potential is limited. To extend proliferation, these cells were previously transduced with viral oncogenes or mouse Bmi-1 + hTERT, but the resultant cell lines did not undergo mucociliary differentiation. We hypothesized that use of human BMI-1 alone would increase the proliferative potential of bronchial epithelial cells while retaining their mucociliary differentiation potential. Cystic fibrosis (CF) and non-CF bronchial epithelial cells were transduced by lentivirus with BMI-1 and then their morphology, replication kinetics, and karyotype were assessed. When differentiated at ALI, mucin production, ciliary function, and transepithelial electrophysiology were measured. Finally, shRNA knockdown of DNAH5 in BMI-1 cells was used to model primary ciliary dyskinesia (PCD). BMI-1-transduced basal cells showed normal cell morphology, karyotype, and doubling times despite extensive passaging. The cell lines underwent mucociliary differentiation when cultured at ALI with abundant ciliation and production of the gel-forming mucins MUC5AC and MUC5B evident. Cilia displayed a normal beat frequency and 9+2 ultrastructure. Electrophysiological characteristics of BMI-1-transduced cells were similar to those of untransduced cells. shRNA knockdown of DNAH5 in BMI-1 cells produced immotile cilia and absence of DNAH5 in the ciliary axoneme as seen in cells from patients with PCD. BMI-1 delayed senescence in bronchial epithelial cells, increasing their proliferative potential but maintaining mucociliary differentiation at ALI. We have shown these cells are amenable to genetic manipulation and can be used to produce novel disease models for research and dissemination.


Assuntos
Brônquios/citologia , Diferenciação Celular , Cílios/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Muco/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Animais , Dineínas do Axonema/metabolismo , Proliferação de Células , Forma Celular , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Dineínas/metabolismo , Impedância Elétrica , Fenômenos Eletrofisiológicos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patologia , Síndrome de Kartagener/fisiopatologia , Cariotipagem , Camundongos , Microtúbulos/metabolismo , Modelos Biológicos , Fenótipo , Transdução Genética
11.
Sci Rep ; 6: 23125, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26975732

RESUMO

Gene therapy for cystic fibrosis using non-viral, plasmid-based formulations has been the subject of intensive research for over two decades but a clinically viable product has yet to materialise in large part due to inefficient transgene expression. Minicircle DNA give enhanced and more persistent transgene expression compared to plasmid DNA in a number of organ systems but has not been assessed in the lung. In this study we compared minicircle DNA with plasmid DNA in transfections of airway epithelial cells. In vitro, luciferase gene expression from minicircles was 5-10-fold higher than with plasmid DNA. In eGFP transfections in vitro both the mean fluorescence intensity and percentage of cells transfected was 2-4-fold higher with minicircle DNA. Administration of equimolar amounts of DNA to mouse lungs resulted in a reduced inflammatory response and more persistent transgene expression, with luciferase activity persisting for 2 weeks from minicircle DNA compared to plasmid formulations. Transfection of equal mass amounts of DNA in mouse lungs resulted in a 6-fold increase in transgene expression in addition to more persistent transgene expression. Our findings have clear implications for gene therapy of airway disorders where plasmid DNA transfections have so far proven inefficient in clinical trials.


Assuntos
Dineínas do Axonema/genética , DNA Circular/genética , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Luciferases de Vaga-Lume/genética , Pulmão/metabolismo , Animais , Dineínas do Axonema/metabolismo , Linhagem Celular , Citocinas/metabolismo , Células Epiteliais/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Luciferases de Vaga-Lume/metabolismo , Camundongos , Plasmídeos , Transfecção , Transgenes
12.
FASEB J ; 29(9): 3876-88, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26062602

RESUMO

Several acute monogenic diseases affect multiple body systems, causing death in childhood. The development of novel therapies for such conditions is challenging. However, improvements in gene delivery technology mean that gene therapy has the potential to treat such disorders. We evaluated the ability of the AAV9 vector to mediate systemic gene delivery after intravenous administration to perinatal mice and late-gestation nonhuman primates (NHPs). Titer-matched single-stranded (ss) and self-complementary (sc) AAV9 carrying the green fluorescent protein (GFP) reporter gene were intravenously administered to fetal and neonatal mice, with noninjected age-matched mice used as the control. Extensive GFP expression was observed in organs throughout the body, with the epithelial and muscle cells being particularly well transduced. ssAAV9 carrying the WPRE sequence mediated significantly more gene expression than its sc counterpart, which lacked the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence. To examine a realistic scale-up to larger models or potentially patients for such an approach, AAV9 was intravenously administered to late-gestation NHPs by using a clinically relevant protocol. Widespread systemic gene expression was measured throughout the body, with cellular tropisms similar to those observed in the mouse studies and no observable adverse events. This study confirms that AAV9 can safely mediate systemic gene delivery in small and large animal models and supports its potential use in clinical systemic gene therapy protocols.


Assuntos
Dependovirus , Feto , Vetores Genéticos , Proteínas de Fluorescência Verde , Transdução Genética/métodos , Tropismo Viral , Animais , Feminino , Feto/citologia , Feto/embriologia , Feto/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Vetores Genéticos/farmacologia , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Haplorrinos , Camundongos , Gravidez
13.
J Immunol Res ; 2015: 397879, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25759840

RESUMO

Following fetal or neonatal gene transfer in mice and other species immune tolerance of the transgenic protein is frequently observed; however the underlying mechanisms remain largely undefined. In this study fetal and neonatal BALB/c mice received adenovirus vector to deliver human factor IX (hFIX) cDNA. The long-term tolerance of hFIX was robust in the face of immune challenge with hFIX protein and adjuvant but was eliminated by simultaneous administration of anti-CD25+ antibody. Naive irradiated BALB/c mice which had received lymphocytes from donors immunised with hFIX developed anti-hFIX antibodies upon immune challenge. Cotransplantation with CD4+CD25+ cells isolated from neonatally tolerized donors decreased the antibody response. In contrast, cotransplantation with CD4+CD25- cells isolated from the same donors increased the antibody response. These data provide evidence that immune tolerance following perinatal gene transfer is maintained by a CD4+CD25+ regulatory population.


Assuntos
Adenoviridae/genética , Fator IX/genética , Fator IX/imunologia , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Tolerância Imunológica , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Anticorpos/sangue , Anticorpos/imunologia , Antígenos CD4/metabolismo , Fator IX/metabolismo , Expressão Gênica , Vetores Genéticos/administração & dosagem , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Depleção Linfocítica , Camundongos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Distribuição Tecidual
14.
Nat Commun ; 6: 6094, 2015 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-25615415

RESUMO

T-cell acute lymphoblastic leukaemias (T-ALL) are aggressive malignant proliferations characterized by high relapse rates and great genetic heterogeneity. TAL1 is amongst the most frequently deregulated oncogenes. Yet, over half of the TAL1(+) cases lack TAL1 lesions, suggesting unrecognized (epi)genetic deregulation mechanisms. Here we show that TAL1 is normally silenced in the T-cell lineage, and that the polycomb H3K27me3-repressive mark is focally diminished in TAL1(+) T-ALLs. Sequencing reveals that >20% of monoallelic TAL1(+) patients without previously known alterations display microinsertions or RAG1/2-mediated episomal reintegration in a single site 5' to TAL1. Using 'allelic-ChIP' and CrispR assays, we demonstrate that such insertions induce a selective switch from H3K27me3 to H3K27ac at the inserted but not the germline allele. We also show that, despite a considerable mechanistic diversity, the mode of oncogenic TAL1 activation, rather than expression levels, impact on clinical outcome. Altogether, these studies establish site-specific epigenetic desilencing as a mechanism of oncogenic activation.


Assuntos
Alelos , Regulação Leucêmica da Expressão Gênica , Proteínas do Grupo Polycomb/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Acetilação , Adulto , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Loci Gênicos , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Células Jurkat , Metilação , Dados de Sequência Molecular , Mutagênese Insercional , Proteínas Nucleares/metabolismo , Plasmídeos/genética , Proteínas do Grupo Polycomb/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Análise de Sobrevida , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Resultado do Tratamento
15.
Methods Mol Biol ; 891: 85-107, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22648769

RESUMO

Vectors derived from the Retroviridae family have several attributes required for successful gene delivery. Retroviral vectors have an adequate payload size for the coding regions of most genes; they are safe to handle and simple to produce. These vectors can be manipulated to target different cell types with low immunogenicity and can permanently insert genetic information into the host cells' genome. Retroviral vectors have been used in gene therapy clinical trials and successfully applied experimentally in vitro, in vivo, and in utero.


Assuntos
Terapia Genética/métodos , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Cuidado Pré-Natal/métodos , Retroviridae/genética , Ensaio de Imunoadsorção Enzimática , Humanos , Reação em Cadeia da Polimerase , Titulometria
16.
Mol Ther ; 20(8): 1610-21, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22547151

RESUMO

Mucopolysaccharidosis IIIA (MPS IIIA or Sanfilippo disease) is a neurodegenerative disorder caused by a deficiency in the lysosomal enzyme sulfamidase (SGSH), catabolizing heparan sulfate (HS). Affected children present with severe behavioral abnormalities, sleep disturbances, and progressive neurodegeneration, leading to death in their second decade. MPS I, a similar neurodegenerative disease accumulating HS, is treated successfully with hematopoietic stem cell transplantation (HSCT) but this treatment is ineffectual for MPS IIIA. We compared HSCT in MPS IIIA mice using wild-type donor cells transduced ex vivo with lentiviral vector-expressing SGSH (LV-WT-HSCT) versus wild-type donor cell transplant (WT-HSCT) or lentiviral-SGSH transduced MPS IIIA cells (LV-IIIA-HSCT). LV-WT-HSCT results in 10% of normal brain enzyme activity, near normalization of brain HS and GM2 gangliosides, significant improvements in neuroinflammation and behavioral correction. Both WT-HSCT and LV-IIIA-HSCT mediated improvements in GM2 gangliosides and neuroinflammation but were less effective at reducing HS or in ameliorating abnormal HS sulfation and had no significant effect on behavior. This suggests that HS may have a more significant role in neuropathology than neuroinflammation or GM2 gangliosides. These data provide compelling evidence for the efficacy of gene therapy in conjunction with WT-HSCT for neurological correction of MPS IIIA where conventional transplant is ineffectual.


Assuntos
Terapia Genética/métodos , Células-Tronco Hematopoéticas/fisiologia , Mucopolissacaridoses/patologia , Mucopolissacaridoses/terapia , Animais , Feminino , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Imuno-Histoquímica , Camundongos
17.
Methods Enzymol ; 507: 29-57, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22365768

RESUMO

Retroviruses are useful tools for the efficient delivery of genes to mammalian cells, owing to their ability to stably integrate into the host cell genome. Over the past few decades, retroviral vectors have been used in gene therapy clinical trials for the treatment of a number of inherited diseases and cancers. The earliest retrovirus vectors were based on simple oncogenic gammaretroviruses such as Moloney murine leukemia virus (MMLV) which, when pseudotyped with envelope proteins from other viruses such as the gibbon ape leukemia virus envelope protein (GALV) or vesicular stomatitis virus G protein (VSV-G), can efficiently introduce genes to a wide range of host cells. However, gammaretroviral vectors have the disadvantage that they are unable to efficiently transduce nondividing or slowly dividing cells. As a result, specific protocols have been developed to activate cells through the use of growth factors and cytokines. In the case of hematopoietic stem cells, activation has to be carefully controlled so that pluripotency is maintained. For many applications, gammaretroviral vectors are being superseded by lentiviral vectors based on human immunodeficiency virus type-1 (HIV-1) which has additional accessory proteins that enable integration in the absence of cell division. In addition, retroviral and lentiviral vector design has evolved to address a number of safety concerns. These include separate expression of the viral genes in trans to prevent recombination events leading to the generation of replication-competent viruses. Further, the development of self-inactivating (SIN) vectors reduces the potential for transactivation of neighboring genes and allows the incorporation of regulatory elements that may target gene expression more physiologically to particular cell types.


Assuntos
Gammaretrovirus/genética , Lentivirus/genética , Técnicas de Cultura de Células , Clonagem Molecular , Gammaretrovirus/isolamento & purificação , Gammaretrovirus/fisiologia , Técnicas de Transferência de Genes , Genes Virais , Engenharia Genética , Marcadores Genéticos , Terapia Genética/métodos , Vetores Genéticos , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Células-Tronco Hematopoéticas/virologia , Humanos , Lentivirus/isolamento & purificação , Lentivirus/fisiologia , Regiões Promotoras Genéticas , Carga Viral , Tropismo Viral
18.
Stem Cells ; 30(6): 1134-43, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22367629

RESUMO

Human mesenchymal stem cells (hMSCs) have been shown to have potential in regenerative approaches in bone and blood. Most protocols rely on their in vitro expansion prior to clinical use. However, several groups including our own have shown that hMSCs lose proliferation and differentiation ability with serial passage in culture, limiting their clinical applications. Cellular prion protein (PrP) has been shown to enhance proliferation and promote self-renewal of hematopoietic, mammary gland, and neural stem cells. Here we show, for the first time, that expression of PrP decreased in hMSC following ex vivo expansion. When PrP expression was knocked down, hMSC showed significant reduction in proliferation and differentiation. In contrast, hMSC expanded in the presence of small molecule 3/689, a modulator of PrP expression, showed retention of PrP expression with ex vivo expansion and extended lifespan up to 10 population doublings. Moreover, cultures produced a 300-fold increase in the number of cells generated. These cells showed a 10-fold increase in engraftment levels in bone marrow 5 weeks post-transplant. hMSC treated with 3/689 showed enhanced protection from DNA damage and enhanced cell cycle progression, in line with data obtained by gene expression profiling. Moreover, upregulation of superoxide dismutase-2 (SOD2) was also observed in hMSC expanded in the presence of 3/689. The increase in SOD2 was dependent on PrP expression and suggests increased scavenging of reactive oxygen species as mechanism of action. These data point to PrP as a good target for chemical intervention in stem cell regenerative medicine.


Assuntos
Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Príons/biossíntese , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Células Cultivadas , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Lentivirus/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Príons/genética , Transfecção
19.
Sci Transl Med ; 3(97): 97ra79, 2011 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-21865537

RESUMO

X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the common cytokine receptor γ chain. These mutations classically lead to complete absence of functional T and natural killer cell lineages as well as to intrinsically compromised B cell function. Although human leukocyte antigen (HLA)-matched hematopoietic stem cell transplantation (HSCT) is highly successful in SCID-X1 patients, HLA-mismatched procedures can be associated with prolonged immunodeficiency, graft-versus-host disease, and increased overall mortality. Here, 10 children were treated with autologous CD34(+) hematopoietic stem and progenitor cells transduced with a conventional gammaretroviral vector. The patients did not receive myelosuppressive conditioning and were monitored for immunological recovery after cell infusion. All patients were alive after a median follow-up of 80 months (range, 54 to 107 months), and a functional polyclonal T cell repertoire was restored in all patients. Humoral immunity only partially recovered but was sufficient in some patients to allow for withdrawal of immunoglobulin replacement; however, three patients developed antibiotic-responsive acute pulmonary infection after discontinuation of antibiotic prophylaxis and/or immunoglobulin replacement. One patient developed acute T cell acute lymphoblastic leukemia because of up-regulated expression of the proto-oncogene LMO-2 from insertional mutagenesis, but maintained a polyclonal T cell repertoire through chemotherapy and entered remission. Therefore, gene therapy for SCID-X1 without myelosuppressive conditioning effectively restored T cell immunity and was associated with high survival rates for up to 9 years. Further studies using vectors designed to limit mutagenesis and strategies to enhance B cell reconstitution are warranted to define the role of this treatment modality alongside conventional HSCT for SCID-X1.


Assuntos
Terapia Genética/métodos , Linfócitos T/imunologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Antígenos CD34/genética , Antígenos CD34/metabolismo , Pré-Escolar , Feminino , Gammaretrovirus/genética , Vetores Genéticos/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Lactente , Masculino , Proto-Oncogene Mas , Transplante de Células-Tronco/métodos , Transplante Autólogo/métodos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/metabolismo
20.
Mol Ther ; 19(11): 2031-9, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21862999

RESUMO

Vector-associated side effects in clinical gene therapy have provided insights into the molecular mechanisms of hematopoietic regulation in vivo. Surprisingly, many retrovirus insertion sites (RIS) present in engrafted cells have been found to cluster nonrandomly in close association with specific genes. Our data demonstrate that these genes directly influence the in vivo fate of hematopoietic cell clones. Analysis of insertions thus far has been limited to individual clinical studies. Here, we studied >7,000 insertions retrieved from various studies. More than 40% of all insertions found in engrafted gene-modified cells were clustered in the same genomic areas covering only 0.36% of the genome. Gene classification analyses displayed significant overrepresentation of genes associated with hematopoietic functions and relevance for cell growth and survival in vivo. The similarity of insertion distributions indicates that vector insertions in repopulating cells cluster in predictable patterns. Thus, insertion analyses of preclinical in vitro and murine in vivo studies as well as vector insertion repertoires in clinical trials yielded concerted results and mark a small number of interesting genomic loci and genes that warrants further investigation of the biological consequences of vector insertions.


Assuntos
Gammaretrovirus/genética , Terapia Genética/efeitos adversos , Vetores Genéticos/efeitos adversos , Genoma , Integração Viral , Animais , Mapeamento Cromossômico , Redes Reguladoras de Genes , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Primatas , Transplantes , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia
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