RESUMO
Specific mutations in the human gene encoding the Wiskott-Aldrich syndrome protein (WASp) that compromise normal auto-inhibition of WASp result in unregulated activation of the actin-related protein 2/3 complex and increased actin polymerizing activity. These activating mutations are associated with an X-linked form of neutropenia with an intrinsic failure of myelopoiesis and an increase in the incidence of cytogenetic abnormalities. To study the underlying mechanisms, active mutant WASp(I294T) was expressed by gene transfer. This caused enhanced and delocalized actin polymerization throughout the cell, decreased proliferation, and increased apoptosis. Cells became binucleated, suggesting a failure of cytokinesis, and micronuclei were formed, indicative of genomic instability. Live cell imaging demonstrated a delay in mitosis from prometaphase to anaphase and confirmed that multinucleation was a result of aborted cytokinesis. During mitosis, filamentous actin was abnormally localized around the spindle and chromosomes throughout their alignment and separation, and it accumulated within the cleavage furrow around the spindle midzone. These findings reveal a novel mechanism for inhibition of myelopoiesis through defective mitosis and cytokinesis due to hyperactivation and mislocalization of actin polymerization.
Assuntos
Actinas/metabolismo , Citocinese , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Mitose , Neutropenia/metabolismo , Neutropenia/patologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Aberrações Cromossômicas , Cromossomos Humanos , Citocinese/efeitos dos fármacos , DNA , Depsipeptídeos/farmacologia , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Mitose/efeitos dos fármacos , Proteínas Mutantes/metabolismo , Poliploidia , Proteínas Recombinantes de Fusão/metabolismo , TransgenesRESUMO
Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.
Assuntos
Códon/genética , Fator VIII/genética , Terapia Genética/métodos , Hemofilia A/terapia , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Ensaio de Imunoadsorção Enzimática , Fator VIII/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Células HEK293 , Hemofilia A/sangue , Hemofilia A/genética , Humanos , Injeções Intravenosas , Lentivirus/genética , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Vírus Formadores de Foco no Baço/genéticaRESUMO
Retroviral and lentiviral vector integration into host-cell chromosomes carries with it a finite chance of causing insertional mutagenesis. This risk has been highlighted by the induction of malignancy in mouse models, and development of lymphoproliferative disease in three individuals with severe combined immunodeficiency-X1 (refs. 2,3). Therefore, a key challenge for clinical therapies based on retroviral vectors is to achieve stable transgene expression while minimizing insertional mutagenesis. Recent in vitro studies have shown that integration-deficient lentiviral vectors can mediate stable transduction. With similar vectors, we now show efficient and sustained transgene expression in vivo in rodent ocular and brain tissues. We also show substantial rescue of clinically relevant rodent models of retinal degeneration. Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. For therapeutic application to postmitotic tissues, this system substantially reduces the risk of insertional mutagenesis.
Assuntos
Terapia Genética/métodos , Vetores Genéticos/genética , Lentivirus/genética , Animais , Encéfalo/citologia , Proteínas de Transporte , Eletrorretinografia , Proteínas do Olho/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Camundongos , Epitélio Pigmentado Ocular/citologia , Ratos , Retina/citologia , Células Tumorais Cultivadas , Integração Viral/genética , cis-trans-IsomerasesRESUMO
X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene encoding the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase catalytic subunit gp91(phox). A recent clinical trial for X-CGD using a spleen focus-forming virus (SFFV)-based γ-retroviral vector has demonstrated clear therapeutic benefits in several patients although complicated by enhancer-mediated mutagenesis and diminution of effectiveness over time due to silencing of the viral long terminal repeat (LTR). To improve safety and efficacy, we have designed a lentiviral vector that directs transgene expression primarily in myeloid cells. To this end, we created a synthetic chimeric promoter that contains binding sites for myeloid transcription factors CAAT box enhancer-binding family proteins (C/EBPs) and PU.1, which are highly expressed during granulocytic differentiation. As predicted, the chimeric promoter regulated higher reporter gene expression in myeloid than in nonmyeloid cells, and in human hematopoietic progenitors upon granulocytic differentiation. In a murine model of stem cell gene therapy for X-CGD, the chimeric vector resulted in high levels of gp91(phox) expression in committed myeloid cells and granulocytes, and restored normal NADPH-oxidase activity. These findings were recapitulated in human neutrophils derived from transduced X-CGD CD34(+) cells in vivo, and suggest that the chimeric promoter will have utility for gene therapy of myeloid lineage disorders such as CGD.
Assuntos
Catepsina G/genética , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/terapia , Células Mieloides/fisiologia , Proteínas Proto-Oncogênicas c-fes/genética , Proteínas Recombinantes de Fusão/genética , Transgenes , Animais , Sequência de Bases , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Genes Ligados ao Cromossomo X , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Granulócitos/metabolismo , Doença Granulomatosa Crônica/enzimologia , Doença Granulomatosa Crônica/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Camundongos , Dados de Sequência Molecular , Mutagênese/genética , Células Mieloides/metabolismo , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Retroviridae/genética , Retroviridae/metabolismo , Vírus Formadores de Foco no Baço/genética , Vírus Formadores de Foco no Baço/metabolismo , Células-Tronco/metabolismo , Sequências Repetidas Terminais , Transativadores/metabolismoRESUMO
The Wiskott-Aldrich syndrome protein (WASp) is a key cytoskeletal regulator in hematopoietic cells. Covalent modification of a conserved tyrosine by phosphorylation has emerged as an important potential determinant of activity, although the physiological significance remains uncertain. In a murine knockin model, mutation resulting in inability to phosphorylate Y293 (Y293F) mimicked many features of complete WASp-deficiency. Although a phosphomimicking mutant Y293E conferred enhanced actin-polymerization, the cellular phenotype was similar due to functional dysregulation. Furthermore, steady-state levels of Y293E-WASp were markedly reduced compared to wild-type WASp and Y293F-WASp, although partially recoverable by treatment of cells with proteasome inhibitors. Consequently, tyrosine phosphorylation plays a critical role in normal activation of WASp in vivo, and is indispensible for multiple tasks including proliferation, phagocytosis, chemotaxis, and assembly of adhesion structures. Furthermore, it may target WASp for proteasome-mediated degradation, thereby providing a default mechanism for self-limiting stimulation of the Arp2/3 complex.
Assuntos
Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Substituição de Aminoácidos , Animais , Sítios de Ligação/genética , Células COS , Linhagem Celular , Movimento Celular , Chlorocebus aethiops , Hematopoese , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Fagocitose , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tirosina/química , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/química , Proteína da Síndrome de Wiskott-Aldrich/genéticaRESUMO
X-linked SCID (SCID-X1) is amenable to correction by gene therapy using conventional gammaretroviral vectors. Here, we describe the occurrence of clonal T cell acute lymphoblastic leukemia (T-ALL) promoted by insertional mutagenesis in a completed gene therapy trial of 10 SCID-X1 patients. Integration of the vector in an antisense orientation 35 kb upstream of the protooncogene LIM domain only 2 (LMO2) caused overexpression of LMO2 in the leukemic clone. However, leukemogenesis was likely precipitated by the acquisition of other genetic abnormalities unrelated to vector insertion, including a gain-of-function mutation in NOTCH1, deletion of the tumor suppressor gene locus cyclin-dependent kinase 2A (CDKN2A), and translocation of the TCR-beta region to the STIL-TAL1 locus. These findings highlight a general toxicity of endogenous gammaretroviral enhancer elements and also identify a combinatorial process during leukemic evolution that will be important for risk stratification and for future protocol design.
Assuntos
Cromossomos Humanos X , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/etiologia , Imunodeficiência Combinada Severa/terapia , Proteínas Adaptadoras de Transdução de Sinal , Antineoplásicos/farmacologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Proteínas de Ligação a DNA/genética , Seguimentos , Humanos , Lactente , Proteínas com Domínio LIM , Masculino , Metaloproteínas/genética , Modelos Biológicos , Mutagênese , Leucemia-Linfoma Linfoblástico de Células T Precursoras/complicações , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Proteínas Proto-Oncogênicas , Receptor Notch1/genética , Receptores de Interleucina-2/genética , Imunodeficiência Combinada Severa/complicaçõesRESUMO
We treated 10 children with X-linked SCID (SCID-X1) using gammaretrovirus-mediated gene transfer. Those with sufficient follow-up were found to have recovered substantial immunity in the absence of any serious adverse events up to 5 years after treatment. To determine the influence of vector integration on lymphoid reconstitution, we compared retroviral integration sites (RISs) from peripheral blood CD3(+) T lymphocytes of 5 patients taken between 9 and 30 months after transplantation with transduced CD34(+) progenitor cells derived from 1 further patient and 1 healthy donor. Integration occurred preferentially in gene regions on either side of transcription start sites, was clustered, and correlated with the expression level in CD34(+) progenitors during transduction. In contrast to those in CD34(+) cells, RISs recovered from engrafted CD3(+) T cells were significantly overrepresented within or near genes encoding proteins with kinase or transferase activity or involved in phosphorus metabolism. Although gross patterns of gene expression were unchanged in transduced cells, the divergence of RIS target frequency between transduced progenitor cells and post-thymic T lymphocytes indicates that vector integration influences cell survival, engraftment, or proliferation.
Assuntos
Complexo CD3 , Gammaretrovirus , Vetores Genéticos , Transplante de Células-Tronco Hematopoéticas , Linfócitos T/imunologia , Integração Viral , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Adulto , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Criança , Pré-Escolar , Feminino , Seguimentos , Sobrevivência de Enxerto/genética , Sobrevivência de Enxerto/imunologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Lactente , Masculino , Transdução Genética , Transplante Autólogo , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologiaAssuntos
Agamaglobulinemia/história , Doenças Genéticas Ligadas ao Cromossomo X/história , Proteínas Tirosina Quinases/história , Proto-Oncogenes , Quinases da Família src/classificação , Tirosina Quinase da Agamaglobulinemia , Agamaglobulinemia/enzimologia , Sequência de Aminoácidos , Linfócitos B/enzimologia , Linhagem da Célula , Mapeamento Cromossômico , Cromossomos Humanos X/genética , Clonagem Molecular , Sequência Consenso , Feminino , Triagem de Portadores Genéticos , Doenças Genéticas Ligadas ao Cromossomo X/enzimologia , História do Século XX , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/classificação , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Inativação do Cromossomo XRESUMO
Wiskott-Aldrich syndrome (WAS) is an X-linked hematological disease characterized by immunodeficiency, eczema, and thrombocytopaenia, and shows promise for treatment with hematopoietic stem cell gene therapy. The immunopathology of WAS is attributable at least in part to defects of cell migration and localization as a result of chemotactic, adhesive, and chemokinetic defects. Whereas previous studies using either gammaretroviral or lentiviral vectors have demonstrated variable correction of T-cell proliferation and dendritic cell (DC) cytoarchitecture, we have used a lentiviral vector expressing an eGFP-WASp fusion protein to test the potential for restoration of cell migratory defects. Multilineage expression of the fusion transgene was present for up to 10 months after primary engraftment, and also in secondary recipients analyzed after a further 9 months. Transduced bone marrow-derived dendritic cells (BMDCs) demonstrated recovery of podosome numbers and turnover, while B cells, BMDCs, and Langerhans cells (LCs) exhibited enhanced chemotactic responses to specific stimuli. As an indication of functionality in vivo, splenic marginal zone B cells and a cutaneous contact hypersensitivity (CHS) response to dinitrofluorobenzene (DNFB) were both partially restored. These proof of principle experiments demonstrate that WAS protein (WASp) transgene expression can be successfully maintained long term in primary and secondary recipients, and that it is associated with a significant repair of migratory defects.
Assuntos
Terapia Genética/métodos , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Animais , Movimento Celular , Transplante de Células , Quimiotaxia , Células Dendríticas/citologia , Dinitrofluorbenzeno/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Células de Langerhans/citologia , Lentivirus/genética , Camundongos , Camundongos Knockout , Modelos Biológicos , BaçoRESUMO
Gene therapy for X-linked severe combined immunodeficiency (SCID-X1) has proven highly effective for long-term restoration of immunity in human subjects. However, the development of lymphoproliferative complications due to dysregulated proto-oncogene expression has underlined the necessity for developing safer vector systems. To reduce the potential for insertional mutagenesis, we have evaluated the efficacy of self-inactivating (SIN) gammaretroviral vectors in cellular and in vivo models of SCID-X1. Vectors incorporating an internal human elongation factor-1alpha regulatory element were capable of fully restoring the lymphoid differentiation potential of gammac-deficient lineage negative cells. Multilineage lymphoid reconstitution of a murine model was achieved at a similar level to that achieved by a conventional long-terminal repeat (LTR)-regulated vector used in previous clinical trials. Functional proliferative responses to mitogenic stimuli were also restored, and serum immunoglobulin levels were normalized. The reduced mutagenic potential conferred by SIN vector configurations and alternative non-LTR-based regulatory elements, together with proven efficacy in correction of cellular defects provides an important platform for development of the next phase of clinical trials for SCID-X1.
Assuntos
Gammaretrovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Northern Blotting , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Reação em Cadeia da Polimerase , Proto-Oncogene Mas , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologiaRESUMO
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 , TransgenesAssuntos
Proteína Morfogenética Óssea 4/genética , Células-Tronco Embrionárias/citologia , RNA Interferente Pequeno/administração & dosagem , Animais , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células-Tronco Embrionárias/efeitos dos fármacos , Humanos , Camundongos , RNA Interferente Pequeno/genética , Regulação para CimaRESUMO
BACKGROUND: X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the common cytokine-receptor gamma chain (gamma(c)), resulting in disruption of development of T lymphocytes and natural-killer cells. B-lymphocyte function is also intrinsically compromised. Allogeneic bone-marrow transplantation is successful if HLA-matched family donors are available, but HLA-mismatched procedures are associated with substantial morbidity and mortality. We investigated the application of somatic gene therapy by use of a gibbon-ape-leukaemia-virus pseudotyped gammaretroviral vector. METHODS: Four children with SCID-X1 were enrolled. Autologous CD34-positive haemopoietic bone-marrow stem cells were transduced ex vivo and returned to the patients without preceding cytoreductive chemotherapy. The patients were monitored for integration and expression of the gamma(c) vector and for functional immunological recovery. FINDINGS: All patients have shown substantial improvements in clinical and immunological features, and prophylactic medication could be withdrawn in two. No serious adverse events have been recorded. T cells responded normally to mitogenic and antigenic stimuli, and the T-cell-receptor (TCR) repertoire was highly diverse. Where assessable, humoral immunity, in terms of antibody production, was also restored and associated with increasing rates of somatic mutation in immunoglobulin genes. INTERPRETATION: Gene therapy for SCID-X1 is a highly effective strategy for restoration of functional cellular and humoral immunity.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/terapia , Terapia Genética , Imunodeficiência Combinada Severa/terapia , Antígenos CD34/análise , Células da Medula Óssea/imunologia , Transplante de Medula Óssea , Pré-Escolar , Gammaretrovirus , Técnicas de Transferência de Genes , Doenças Genéticas Ligadas ao Cromossomo X/genética , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos , Humanos , Imunidade , Imunoglobulinas/sangue , Lactente , Subunidade gama Comum de Receptores de Interleucina , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Mutação , Receptores de Interleucina-7/genética , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Linfócitos T/imunologia , Transdução GenéticaRESUMO
Prolonged exposure of human hematopoietic stem cells (HSC) to growth factors for efficient transduction by murine oncoretroviral vectors has major detrimental effects on repopulating activity. In this study, we have used a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped human immunodeficiency virus type 1 (HIV-1) lentiviral-based vector system to transduce cord blood (CB) CD34+ cells over a limited time period (< or =24 hours). Under these conditions, significant gene marking was observed in engrafted human lymphoid, myeloid, and progenitor cells in all transplanted Severe Combined Immunodeficient (SCID) mice. To enhance the level of gene expression in hematopoietic cells, we also generated a series of lentiviral vectors incorporating the spleen focus forming virus (SFFV) long terminal repeat (LTR) sequences, and the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). By including the central polypurine tract (cPPT) sequence of HIV-1 we were then able to achieve high levels of transduction (over 80%) and gene expression in vivo after a single exposure to viral supernatant. These results demonstrate that lentiviral vectors are highly effective for gene transfer to human HSC, and that SFFV regulatory sequences can be successfully incorporated to enhance the long-term expression of a transgene in primary human hematopoietic cells in vivo.
Assuntos
Elementos Facilitadores Genéticos , Técnicas de Transferência de Genes , Vetores Genéticos , HIV-1/genética , Lentivirus/genética , Glicoproteínas de Membrana , Animais , Antígenos CD34/biossíntese , Células Cultivadas , Citocinas/biossíntese , Sangue Fetal/metabolismo , Citometria de Fluxo , Proteínas de Fluorescência Verde , Células-Tronco Hematopoéticas/metabolismo , Vírus da Hepatite B da Marmota/genética , Humanos , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos SCID , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Purinas/química , Vírus Formadores de Foco no Baço/genética , Sequências Repetidas Terminais , Transdução Genética , Proteínas do Envelope Viral/genéticaRESUMO
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/metabolismoRESUMO
Genetic defects in the purine salvage enzyme adenosine deaminase (ADA) lead to severe combined immunodeficiency (SCID) with profound depletion of T, B, and natural killer cell lineages. Human leukocyte antigen-matched allogeneic hematopoietic stem cell transplantation (HSCT) offers a successful treatment option. However, individuals who lack a matched donor must receive mismatched transplants, which are associated with considerable morbidity and mortality. Enzyme replacement therapy (ERT) for ADA-SCID is available, but the associated suboptimal correction of immunological defects leaves patients susceptible to infection. Here, six children were treated with autologous CD34-positive hematopoietic bone marrow stem and progenitor cells transduced with a conventional gammaretroviral vector encoding the human ADA gene. All patients stopped ERT and received mild chemotherapy before infusion of gene-modified cells. All patients survived, with a median follow-up of 43 months (range, 24 to 84 months). Four of the six patients recovered immune function as a result of engraftment of gene-corrected cells. In two patients, treatment failed because of disease-specific and technical reasons: Both restarted ERT and remain well. Of the four reconstituted patients, three remained off enzyme replacement. Moreover, three of these four patients discontinued immunoglobulin replacement, and all showed effective metabolic detoxification. All patients remained free of infection, and two cleared problematic persistent cytomegalovirus infection. There were no adverse leukemic side effects. Thus, gene therapy for ADA-SCID is safe, with effective immunological and metabolic correction, and may offer a viable alternative to conventional unrelated donor HSCT.
Assuntos
Adenosina Desaminase/metabolismo , Agamaglobulinemia/terapia , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Imunodeficiência Combinada Severa/terapia , Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Adenosina Desaminase/imunologia , Agamaglobulinemia/imunologia , Pré-Escolar , Feminino , Dosagem de Genes/genética , Vetores Genéticos/genética , Humanos , Lactente , Masculino , Imunodeficiência Combinada Severa/imunologia , Linfócitos T/imunologiaAssuntos
Dinoprostona/biossíntese , Fibroblastos/patologia , Marcação de Genes , Terapia Genética , Integrinas/genética , Isoenzimas/genética , Pulmão/metabolismo , Prostaglandina-Endoperóxido Sintases/genética , Fibrose Pulmonar/terapia , Animais , Divisão Celular , Ciclo-Oxigenase 2 , Vetores Genéticos , Humanos , Técnicas In Vitro , Isoenzimas/biossíntese , Luciferases/genética , Pulmão/patologia , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidiletanolaminas/genética , Plasmídeos/genética , Prostaglandina-Endoperóxido Sintases/biossíntese , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , TransfecçãoRESUMO
BACKGROUND: Primary immunodeficiencies (PID) are a group of inherited diseases that affect the development or activity of the immune system. In severe cases allogeneic haematopoietic stem cell transplantation has proved to be a successful curative modality but it is limited by toxicity and reduced efficacy in mismatched donor settings. OBJECTIVE: Gene therapy for PID has been developed as an alternative strategy and has entered the clinical arena. In this review we discuss the outcomes of recent gene therapy trials and some of the problems that remain to be tackled. METHODS: Results from clinical trials for X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficient SCID (ADA-SCID), and X-linked chronic granulomatous disease (X-CGD) are discussed. In addition, other conditions are highlighted such as the Wiskott Aldrich Syndrome (WAS) for which gene therapy has shown considerable promise in preclinical studies, and are currently being translated into novel clinical approaches. RESULTS/CONCLUSION: Whilst these encouraging results demonstrate that gene therapy can be used successfully to treat monogenic PID, the occurrence of vector-related side effects has highlighted the need for accurate assessment of the associated risks and a requirement for improvements in vector design.
Assuntos
Terapia Genética , Síndromes de Imunodeficiência/terapia , Animais , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/terapia , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Retroviridae/genética , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/terapia , Resultado do Tratamento , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapiaRESUMO
The transforming growth factor-beta-related factor bone morphogenetic protein 4 (BMP4) is expressed in the human embryonic aorta-gonad-mesonephros (AGM) coincident with the emergence of haematopoietic cells and influences postnatal mammalian haematopoietic stem cells in vitro. To investigate the role of BMP4 in mammalian embryonic haematopoiesis, cells were isolated from murine AGM and two populations of CD34(+) cells with different levels of c-Kit expression and multipotency were identified. CD34(+)/c-Kit(high) cells express CD45 and are haematopoietic-restricted progenitors. In contrast, CD34(+)/c-Kit(low) cells are Flk1+/CD45(neg) and generate adherent colonies in ex vivo culture that resemble haemangioblast colonies identified in other systems. The addition of BMP4 to AGM cells resulted in expansion of the CD34(+)/c-Kit(low) cell pool within 48 h, via a combination of down modulation of the c-Kit receptor in CD34(+)/c-Kit(high) cells and proliferation. In long-term culture, BMP4 increased the growth/survival of CD34(+)/c-Kit(high) haematopoietic progenitors, effects that were blocked by BMP inhibitors. CD34(+)/c-Kit(high) progenitors cultured with BMP4 also generated adherent colonies typical of c-Kit(low) cells. These results suggest that BMP4 regulates c-Kit expression and differentiation potential in CD34(+) AGM cells and supports a role for BMP signalling in the maintenance of multipotency during embryonic haematopoiesis, providing an insight into stem cell homeostasis within the mammalian haematopoietic niche.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/fisiologia , Proteínas Proto-Oncogênicas c-kit/genética , Animais , Antígenos CD34/análise , Aorta/embriologia , Proteína Morfogenética Óssea 4 , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas de Transporte/farmacologia , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Gônadas/embriologia , Mesonefro/fisiologia , Camundongos , Células-Tronco Multipotentes/imunologia , Técnicas de Cultura de TecidosRESUMO
Retroviral transfer of the Herpes Simplex thymidine kinase (HSVTK) suicide gene to donor T cells has been used as a safety strategy against graft-versus-host disease following allogeneic stem cell transplantation. The feasibility of this strategy in human studies has been demonstrated, but a number of limitations have become apparent. Preactivation of donor lymphocytes using mitogens or monoclonal antibodies is essential for retroviral transduction, but can compromise subsequent T-cell function in vivo. We report the application of lentiviral vectors for transduction of T cells in cytokine culture, without activation through the T-cell receptor. Using vectors encoding either enhanced green fluorescent protein or a truncated CD34/mutant HSVTK fusion selection/suicide construct, we investigated the properties of T cells after gene modification. We found that following cytokine stimulation, a fraction of T cells undergoes division, and transgene expression occurred predominantly in these cells. Antiviral and alloreactive responses were preserved in these populations, and in contrast to fully activated T cells, there was minimal perturbation of regulatory T-cell numbers. We conclude that the use of interleukin-7 for lentiviral transduction offers the greatest potential for gene transfer to T cells without loss of function, and is favored for the clinical production of suicide gene modified T cells.