RESUMO
Viruses have evolved to efficiently express their genes in host cells, which makes them ideally suited as gene delivery vectors for gene and immunotherapies. Replication competent (RC) viral vectors encoding foreign or self-proteins induce strong T-cell responses that can be used for the development of effective cancer treatments. Replication-defective (RD) viral vectors encoding self-proteins are non-immunogenic when introduced in a host naïve for the cognate virus. RD viral vectors can be used to develop gene replacement therapies for genetic disorders and tolerization therapies for autoimmune diseases and allergies. Degenerative/inflammatory diseases are associated with chronic inflammation and immune responses that damage the tissues involved. These diseases therefore strongly resemble autoimmune diseases. This review deals with the use of RC and RD viral vectors for unraveling the pathogenesis of immune-related diseases and their application to the development of the next generation prophylactics and therapeutics for todays' major diseases.
Assuntos
Vetores Genéticos , Vírus , Técnicas de Transferência de Genes , Genes Virais , Terapia GenéticaRESUMO
Viruses efficiently transfer and express their genes in host cells and evolve to evade the host's defense responses. These properties render them highly attractive for use as gene delivery vectors in vaccines, gene, and immunotherapies. Among the viruses used as gene delivery vectors, the macaque polyomavirus Simian Virus 40 (SV40) is unique in its capacity to evade intracellular antiviral defense responses upon cell entry. We here describe the unique way by which SV40 particles deliver their genomes in the nucleus of permissive cells and how they prevent presentation of viral antigens to the host's immune system. The non-immunogenicity in its natural host is not only of benefit to the virus but also to us in developing effective SV40 vector-based treatments for today's major human diseases.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos , Vírus 40 dos Símios , Citoplasma/genética , Citoplasma/imunologia , Humanos , Transporte Proteico/genética , Transporte Proteico/imunologia , Vírus 40 dos Símios/genética , Vírus 40 dos Símios/imunologiaRESUMO
Type 1 diabetes mellitus (T1DM) is due to the selective destruction of islet beta cells by immune cells. Current therapies focused on repressing the immune attack or stimulating beta cell regeneration still have limited clinical efficacy. Therefore, it is timely to identify innovative targets to dampen the immune process, while promoting beta cell survival and function. Liver receptor homologue-1 (LRH-1) is a nuclear receptor that represses inflammation in digestive organs, and protects pancreatic islets against apoptosis. Here, we show that BL001, a small LRH-1 agonist, impedes hyperglycemia progression and the immune-dependent inflammation of pancreas in murine models of T1DM, and beta cell apoptosis in islets of type 2 diabetic patients, while increasing beta cell mass and insulin secretion. Thus, we suggest that LRH-1 agonism favors a dialogue between immune and islet cells, which could be druggable to protect against diabetes mellitus.
Assuntos
Comunicação Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/terapia , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Fenalenos/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Insulina/metabolismo , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/patologia , Transplante das Ilhotas Pancreáticas , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/imunologia , Estreptozocina , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Transplante HeterólogoRESUMO
The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene and characterized by severe thrombocytopenia. Although the role of WASp in terminally differentiated lymphocytes and myeloid cells is well characterized, its role in early hematopoietic differentiation and in platelets (Plts) biology is poorly understood. In the present manuscript, we have used zinc finger nucleases targeted to the WAS locus for the development of two isogenic WAS knockout (WASKO) human embryonic stem cell lines (hESCs). Upon hematopoietic differentiation, hESCs-WASKO generated increased ratios of CD34(+)CD45(+) progenitors with altered responses to stem cell factor compared to hESCs-WT. When differentiated toward the megakaryocytic linage, hESCs-WASKO produced increased numbers of CD34(+)CD41(+) progenitors, megakaryocytes (MKs), and Plts. hESCs-WASKO-derived MKs and Plts showed altered phenotype as well as defective responses to agonist, mimicking WAS patients MKs and Plts defects. Interestingly, the defects were more evident in WASp-deficient MKs than in WASp-deficient Plts. Importantly, ectopic WAS expression using lentiviral vectors restored normal Plts development and MKs responses. These data validate the AND-1_WASKO cell lines as a human cellular model for basic research and for preclinical studies for WAS.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Hematopoéticas/citologia , Megacariócitos/citologia , Modelos Biológicos , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Antígenos CD34/metabolismo , Diferenciação Celular , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Antígenos Comuns de Leucócito/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismoRESUMO
Human embryonic stem cells (hESCs) are a unique in vitro model for studying human developmental biology and represent a potential source for cell replacement strategies. Platelets can be generated from cord blood progenitors and hESCs; however, the molecular mechanisms and determinants controlling the in vitro megakaryocytic specification of hESCs remain elusive. We have recently shown that stem cell leukemia (SCL) overexpression accelerates the emergence of hemato-endothelial progenitors from hESCs and promotes their subsequent differentiation into blood cells with higher clonogenic potential. Given that SCL participates in megakaryocytic commitment, we hypothesized that it may potentiate megakaryopoiesis from hESCs. We show that ectopic SCL expression enhances the emergence of megakaryocytic precursors, mature megakaryocytes (MKs), and platelets in vitro. SCL-overexpressing MKs and platelets respond to different activating stimuli similar to their control counterparts. Gene expression profiling of megakaryocytic precursors shows that SCL overexpression renders a megakaryopoietic molecular signature. Connectivity Map analysis reveals that trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), both histone deacetylase (HDAC) inhibitors, functionally mimic SCL-induced effects. Finally, we confirm that both TSA and SAHA treatment promote the emergence of CD34(+) progenitors, whereas valproic acid, another HDAC inhibitor, potentiates MK and platelet production. We demonstrate that SCL and HDAC inhibitors are megakaryopoiesis regulators in hESCs.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Plaquetas/metabolismo , Células-Tronco Embrionárias/metabolismo , Redes Reguladoras de Genes , Megacariócitos/metabolismo , Proteínas Proto-Oncogênicas/genética , Trombopoese/genética , Antígenos CD34/genética , Antígenos CD34/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Diferenciação Celular , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Megacariócitos/citologia , Megacariócitos/efeitos dos fármacos , Plasmídeos/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Trombopoese/efeitos dos fármacos , Transcrição Gênica , Ácido Valproico/farmacologia , VorinostatRESUMO
Mesenchymal stromal cells (MSCs) represent a promising tool for therapy in regenerative medicine, transplantation, and autoimmune disease due to their trophic and immunomodulatory activities. However, we are still far from understanding the mechanisms of action of MSCs in these processes. Transforming growth factor (TGF)-ß1 is a pleiotropic cytokine involved in MSC migration, differentiation, and immunomodulation. Recently, glycoprotein A repetitions predominant (GARP) was shown to bind latency-associated peptide (LAP)/TGF-ß1 to the cell surface of activated Foxp3(+) regulatory T cells (Tregs) and megakaryocytes/platelets. In this manuscript, we show that human and mouse MSCs express GARP which presents LAP/TGF-ß1 on their cell surface. Silencing GARP expression in MSCs increased their secretion and activation of TGF-ß1 and reduced their proliferative capacity in a TGF-ß1-independent manner. Importantly, we showed that GARP expression on MSCs contributed to their ability to inhibit T-cell responses in vitro. In summary, we have found that GARP is an essential molecule for MSC biology, regulating their immunomodulatory and proliferative activities. We envision GARP as a new target for improving the therapeutic efficacy of MSCs and also as a novel MSC marker.
Assuntos
Proteínas de Membrana/biossíntese , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Humanos , Imunomodulação , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Multiple sclerosis (MS) is a severe debilitating disorder characterized by progressive demyelination and axonal damage of the central nervous system (CNS). Current therapies for MS inhibit the immune response and demonstrate reasonable benefits if applied during the early phase of relapsingremitting MS (RRMS) while there are no treatments for patients that progress neither to the chronic phase nor for the primary progressive form of the disease. In this manuscript, we have studied the therapeutic efficacy of a cell and gene therapy strategy for the treatment of a mouse model of chronic MS [myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE)]. We used allogenic mesenchymal stem cells (MSCs) asa therapeutic tool and also as vehicle to deliver fully processed 3.3-kDa vasoactive intestinal peptide (VIP) to the peripheral immune organs and to the inflamed CNS. Intraperitoneal administrations of MSCs expressing VIP stopped progression and reduced symptoms when administered at peak of disease. The improvement in clinical score correlated with diminished peripheral T-cell responses against MOG as well as lower inflammation,lower demyelination, and higher neuronal integrity in the CNS. Interestingly, neither lentiviral vectors expressing VIP nor unmodified MSCs were therapeutic when administer at the peak of disease. The increased therapeutic effect of MSCs expressing VIP over unmodified MSCs requires the immunoregulatory and neuroprotective roles of both VIP and MSCs and the ability of the MSCs to migrate to peripheral lymph organs and the inflamed CNS.
Assuntos
Encefalomielite Autoimune Experimental/terapia , Células-Tronco Mesenquimais/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Tecido Adiposo/citologia , Animais , Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Feminino , Fatores de Transcrição Forkhead/metabolismo , Imunofenotipagem , Inflamação/patologia , Infusões Parenterais , Lentivirus/genética , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/toxicidade , Linfócitos T/citologia , Linfócitos T/imunologia , Transplante Homólogo , Peptídeo Intestinal Vasoativo/genéticaRESUMO
We reported previously that prostaglandin E2 (PGE2) up-regulates IL-23 in vitro in bone marrow-derived dendritic cells and in vivo in models of collagen-induced arthritis and inflammatory bowel disease, leading to preferential Th17 development and activity. There is very little information on the molecular mechanisms involved in the PGE2-induced up-regulation of Il23a gene expression. In this study we investigated the signaling pathways and transcription factors involved in the stimulatory effect of PGE2. Although PGE2 does not induce IL-23p19 expression by itself, it synergizes with both extra- and intracellular Toll-like receptor ligands and with inflammatory cytokines such as TNFα. We established that the effect of PGE2 in conjunction with either LPS or TNFα is mediated through the EP4 receptor and the cAMP-dependent activation of both protein kinase A (PKA) and exchange protein activated by cAMP (EPAC). Using the EP4 agonist PGE(1)OH in conjunction with TNFα, we found that PKA-induced phosphorylation of cAMP-response element-binding protein ((P)CREB) and EPAC-induced phosphorylation of C/AATT enhancer-binding protein ß ((P)C/EBPß) mediate the stimulatory effect of PGE2 on IL-23p19 expression. This is the first report of CREB and C/EBPß involvement in Il23a promoter activation. Mutation within the putative CREB and C/EBP sites combined with in vivo DNA binding (ChIP) assays identified the distal CREB site (-1125) and the two proximal C/EBP sites (-274 and -232) as essential for PKA-activated CREB and EPAC-activated C/EBPß-induced IL-23p19 expression.
Assuntos
Células da Medula Óssea/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Dendríticas/metabolismo , Dinoprostona/fisiologia , Subunidade p19 da Interleucina-23/metabolismo , Adenilil Ciclases/metabolismo , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Animais , Sítios de Ligação , Células da Medula Óssea/imunologia , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Dendríticas/imunologia , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina , Interleucina-12/genética , Interleucina-12/metabolismo , Subunidade p19 da Interleucina-23/genética , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Ligação Proteica , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Sistemas do Segundo Mensageiro , Receptores Toll-Like/metabolismo , Transcrição Gênica , Fator de Necrose Tumoral alfa/fisiologia , Regulação para CimaRESUMO
Genetic manipulation of human embryonic stem cells (hESCs) is instrumental for tracing lineage commitment and to studying human development. Here we used hematopoietic-specific Wiskott-Aldrich syndrome gene (WAS)-promoter driven lentiviral vectors (LVs) to achieve highly specific gene expression in hESCs-derived hematopoietic cells. We first demonstrated that endogenous WAS gene was not expressed in undifferentiated hESCs but was evident in hemogenic progenitors (CD45(-)CD31(+)CD34(+)) and hematopoietic cells (CD45(+)). Accordingly, WAS-promoter driven LVs were unable to express the eGFP transgene in undifferentiated hESCs. eGFP(+) cells only appeared after embryoid body (EB) hematopoietic differentiation. The phenotypic analysis of the eGFP(+) cells showed marking of different subpopulations at different days of differentiation. At days 10-15, AWE LVs tag hemogenic and hematopoietic progenitors cells (CD45(-)CD31(+)CD34(dim) and CD45(+)CD31(+)CD34(dim)) emerging from hESCs and at day 22 its expression became restricted to mature hematopoietic cells (CD45(+)CD33(+)). Surprisingly, at day 10 of differentiation, the AWE vector also marked CD45(-)CD31(low/-)CD34(-) cells, a population that disappeared at later stages of differentiation. We showed that the eGFP(+)CD45(-)CD31(+) population generate 5 times more CD45(+) cells than the eGFP(-)CD45(-)CD31(+) indicating that the AWE vector was identifying a subpopulation inside the CD45(-)CD31(+) cells with higher hemogenic capacity. We also showed generation of CD45(+) cells from the eGFP(+)CD45(-)CD31(low/-)CD34(-) population but not from the eGFP(-)CD45(-)CD31(low/-)CD34(-) cells. This is, to our knowledge, the first report of a gene transfer vector which specifically labels hemogenic progenitors and hematopoietic cells emerging from hESCs. We propose the use of WAS-promoter driven LVs as a novel tool to studying human hematopoietic development.
Assuntos
Células-Tronco Embrionárias/citologia , Vetores Genéticos , Lentivirus/genética , Regiões Promotoras Genéticas , Sequência de Bases , Linhagem da Célula , Células Cultivadas , Primers do DNA , Citometria de Fluxo , Humanos , Reação em Cadeia da PolimeraseRESUMO
Lentiviral vectors (LVs) are considered one of the most promising vehicles to efficiently deliver genetic information for basic research and gene therapy approaches. Combining LVs with drug-inducible expression systems should allow tight control of transgene expression with minimal side effect on relevant target cells. A new doxycycline-regulated system based on the original TetR repressor was developed in 1998 as an alternative to the TetR-VP16 chimeras (tTA and rtTA) to avoid secondary effects due to the expression of transactivator domains. However, previously described TetR-based systems required cell cloning and/or antibiotic selection of tetracycline-responsive cells in order to achieve good regulation. In the present manuscript we have constructed a dual Tet-ON system based on two lentiviral vectors, one expressing the TetR through the spleen focus forming virus (SFFV) promoter (STetR) and a second expressing eGFP through the regulatable CMV-TetO promoter (CTetOE). Using these vectors we have demonstrated that the TetR repressor, contrary to the reverse transactivator (rtTA), can be expressed in excess to bind and modulate a high number of TetO operons. We have also showed that this dual vector system can generate regulatable bulk cell lines (expressing high levels of TetR) that are able to modulate transgene expression either by varying doxycycline concentration and/or by varying the amount of CTetOE vector genomes per cell. Based on these results we have developed a new all-in-one lentiviral vector (CEST) driving the expression of TetR through the SFFV promoter and the expression of eGFP through the doxycycline-responsive CMV-TetO operon. This vector efficiently produced Tet-ON regulatable immortalized (293T) and primary (human mesenchymal stem cells and human primary fibroblasts) cells. Bulk doxycycline-responsive cell lines express high levels of the transgene with low amount of doxycycline and are phenotypically indistinct from its parental cells.
Assuntos
Técnicas de Cultura de Células/métodos , Técnicas Genéticas , Vetores Genéticos/genética , Lentivirus/genética , Proteínas Repressoras/metabolismo , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/genética , Doxiciclina/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lentivirus/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Regiões Promotoras Genéticas/genética , Transporte Proteico/efeitos dos fármacos , Transgenes/genéticaRESUMO
BACKGROUND: Acute and chronic inflammation play essential roles in inflammatory/autoimmune conditions. Protective anti-inflammatory effects of the n-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were reported in animal models of colitis, sepsis, and stroke. Since dendritic cells (DC) represent the essential cellular link between innate and adaptive immunity and have a prominent role in tolerance for self-antigens, we sought to investigate the impact of DHA on DC maturation and proinflammatory cytokine production. METHODS: Murine bone marrow-derived DC were treated with DHA and stimulated with various toll-like receptor (TLR) ligands. Flow cytometry was used to determine the levels of surface maturation markers and endocytic activity. Cytokine expression and secretion were measured by real-time RT-PCR and ELISA assays. PPARgamma and NFkappaB activity in nuclear extracts were determined by binding to specific oligonucleotide sequences using ELISA-based assays. In vivo effects of DHA were assessed in splenic DC from LPS-inoculated mice maintained on a DHA-enriched diet. RESULTS: DHA maintained the immature phenotype in bone marrow-derived DC by preventing the upregulation of MHCII and costimulatory molecules (CD40, CD80 and CD86) and maintaining high levels of endocytic activity. DHA inhibited the production of pro-inflammatory cytokines, including the IL-12 cytokine family (IL-12p70, IL-23, and IL-27), from DC stimulated with TLR2, 3, 4, and 9 ligands. DHA inhibition of IL-12 expression was mediated through activation of PPARgamma and inhibition of NFkappaBp65 nuclear translocation. DHA exerted a similar inhibitory effect on IL-12 and IL-23 expression in vivo in LPS-inoculated mice maintained on a DHA-enriched diet. CONCLUSIONS: Exposure of bone marrow-derived DC to DHA resulted in the maintenance of an immature phenotype and drastic reduction in proinflammatory cytokine release. DHA inhibited the expression and secretion of the IL-12 cytokine family members (IL-12p70, IL-23 and IL-27), which play essential roles in the differentiation of the proinflammatory Th1/Th17 effector cells. The effect of DHA on IL-12 expression was mediated through activation of PPARgamma and inhibition of NFkappaB. Inhibition of IL-12 and IL-23 expression was also evident in splenic DC from mice fed a DHA-enriched diet, suggesting that dietary DHA acts as an anti-inflammatory agent in vivo.
Assuntos
Células Dendríticas/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Interleucina-12/biossíntese , NF-kappa B/metabolismo , Animais , Doenças Autoimunes/metabolismo , Células da Medula Óssea/citologia , Antígeno CD11c/biossíntese , Citocinas/metabolismo , Células Dendríticas/citologia , Modelos Animais de Doenças , Técnicas In Vitro , Inflamação , Interleucina-23/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Sepse/patologia , Acidente Vascular Cerebral/patologiaRESUMO
Dendritic cells (DCs) initiate immune responses as well as tolerance. We showed previously that the neuropeptide vasoactive intestinal peptide (VIP) suppresses innate immune responses, modulates adaptive responses by generating regulatory T cells (Treg) through the induction of tolerogenic DCs (tDCs), and has therapeutic effects in models of autoimmune/inflammatory disorders. Systemic VIP administration is limited by its short biological half-life and by its pleiotropic effects on the cardiovascular system and gastrointestinal tract. Therefore, we used lentiviral vectors to genetically engineer VIP-expressing bone marrow-derived DC (BMDC) and characterized the transduced LentiVIP-DC in terms of phenotype and therapeutic effects in models of experimental autoimmune encephalomyelitis (EAE) and cecal ligation and puncture (CLP) sepsis. LentiVIP-DCs secrete VIP, and resemble tDCs through lack of co-stimulatory molecule upregulation, lack of proinflammatory cytokine secretion, increased interleukin (IL)-10 production, and poor stimulation of allogeneic T cells. A single inoculation of LentiVIP-DC in EAE or CLP mice had therapeutic effects, which correlated with reduced expression of proinflammatory cytokines and increased IL-10 production in spinal cord and peritoneal fluid, respectively. In contrast to systemic VIP administration that requires repeated, high-dose inoculations, local delivery of VIP by LentiVIP-DC may represent a promising therapeutic tool for the treatment of autoimmune diseases and inflammatory disorders.
Assuntos
Diferenciação Celular/fisiologia , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Transdução Genética/métodos , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Células da Medula Óssea/citologia , Diferenciação Celular/genética , Movimento Celular/genética , Movimento Celular/fisiologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Vetores Genéticos , Lentivirus , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Peptídeo Intestinal Vasoativo/genéticaRESUMO
Abstract The development of vectors that express a therapeutic transgene efficiently and specifically in hematopoietic cells (HCs) is an important goal for gene therapy of hematological disorders. We have previously shown that a 500-bp fragment from the proximal Was gene promoter in a lentiviral vector (LV) was sufficient to achieve more than 100-fold higher levels of Wiskott-Aldrich syndrome protein in HCs than in nonhematopoietic cells (non-HCs). We show now that this differential was reduced up to 10 times when the enhanced green fluorescent protein gene (eGFP) was expressed instead of Was in the same LV backbone. Insertion of Was cDNA sequences downstream of eGFP in these LVs had a negative effect on transgene expression. This effect varied in different cell types but, overall, Was cDNA sequences increased the hematopoietic specificity of Was promoter-driven LV. We have characterized the minimal fragment required to increase hematopoietic specificity and have demonstrated that the mechanism involves Was promoter regulation and RNA processing. In addition, we have shown that Was cDNA sequences interfere with the enhancer activity of the woodchuck posttranscriptional regulatory element. These results represent the first data showing the role of Was intragenic sequences in gene regulation.
Assuntos
Terapia Genética/métodos , Vetores Genéticos/genética , Doenças Hematológicas/terapia , Lentivirus/genética , Regiões Promotoras Genéticas/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Western Blotting , Primers do DNA/genética , DNA Complementar/genética , Citometria de Fluxo , Fluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Doenças Hematológicas/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células Jurkat , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transgenes/genéticaRESUMO
Primary immunodeficiencies (PID) are caused by mutations in genes that impair the development or activity of the immune system. Although bone marrow transplants achieve long time restoration in up to 90% of treated patients, morbidity and mortality are still high for some PID and adequate donors are not always available. Gene Therapy (GT) was envisioned as an alternative treatment for PID by inserting the correct gene into the patient's haematopoietic stem cells (HSCs). Up to date, GT for PID has succeeded in 40 of 44 patients treated in four clinical trials. However, five children enrolled in the SCID-X1 clinical trial developed leukaemia-like disease produced by aberrant expression of oncogenes. This phenomenon resulted fatal in one patient and represented a severe setback for gene therapy. Since then, vector development has been a priority in the GT field, by refining existing Murine laeukemia virus (MLV)-based vectors or by developing new ones. This review summarize existing methodologies for PID GT highlighting the importance of animal models in the PID GT success and focusing on new gene transfer vectors to achieve safe, efficient and stable gene modification.
Assuntos
Terapia Genética/métodos , Vetores Genéticos/efeitos adversos , Síndromes de Imunodeficiência/terapia , Transfecção/métodos , Animais , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Humanos , Lentivirus/genética , Camundongos , Retroviridae/genética , Reparo Gênico Alvo-Dirigido/métodosRESUMO
OBJECTIVE: Vasoactive intestinal peptide (VIP) has been shown to exert potent immunomodulatory activity, and the use of lentiviral vectors has been found to be an effective means of gene delivery. The present study was therefore undertaken to investigate the feasibility and efficiency of gene therapy using lentiviral vectors expressing VIP (LentiVIP) for the treatment of rheumatoid arthritis (RA). METHODS: We evaluated the therapeutic potential of the gene therapy strategy in the collagen-induced arthritis (CIA) mouse model, administering the vectors at different phases of the disease. The inflammatory response was determined by measuring the levels of various inflammatory cytokines and chemokines in the joints and serum. The Th1-mediated response was evaluated by determining the proliferative response and cytokine profile of T cells stimulated with autoantigen. RESULTS: A single intraperitoneal injection of LentiVIP was highly effective in treating CIA. Mice with established, severe arthritis showed complete regression of the disease. The therapeutic effect of LentiVIP was associated with widespread biodistribution of the vector and increased VIP levels, especially in joints and lymphoid organs, and was mediated through a striking reduction of the 2 deleterious components of the disease, i.e., the autoimmune response (self-reactive Th1 cell activity and autoantibody production) and the inflammatory response. LentiVIP treatment also induced the generation and/or activation of CD4+,CD25+,FoxP3+ Treg cells in arthritic mice. CONCLUSION: Our findings show that in vivo administration of lentiviral vector expressing VIP produces one of the most potent therapeutic effects described so far in any animal model of RA. We propose that VIP gene transfer should be further investigated as a potential novel, effective treatment of RA and other chronic autoimmune disorders.
Assuntos
Artrite Experimental/genética , Artrite Experimental/terapia , DNA Complementar/uso terapêutico , Terapia Genética , Peptídeo Intestinal Vasoativo/genética , Animais , Artrite Experimental/metabolismo , Vetores Genéticos/uso terapêutico , Inflamação/terapia , Lentivirus/genética , Camundongos , Linfócitos T Reguladores/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Peptídeo Intestinal Vasoativo/uso terapêuticoRESUMO
Efficient and safe gene modification of hematopoietic stem cells is a requirement for gene therapy of primary immunodeficiencies such as Wiskott-Aldrich syndrome. However, deregulated expression or ectopic expression in the progeny of transduced nonhematopoietic progenitor cells may lead to unwanted toxicity. We therefore analyzed the effect of ectopic expression of Wiskott-Aldrich syndrome protein (WASp) and the potential benefits of hematopoietic-specific lentiviral vectors (driven by the WAS proximal promoter). Overexpression of WASp by constitutive lentiviral vectors is highly toxic in nonhematopoietic cells because it causes dramatic changes in actin localization and polymerization that result in decreased cell viability, as evidenced by a significant growth disadvantage of WASp-overexpressing nonhematopoietic cells and increased cell death. These toxic effects do not affect cells of hematopoietic origin because, remarkably, we found that WASp cannot be readily overexpressed in T cells, even after multiple vector integrations per cell. The adverse cellular effects found after transduction of nonhematopoietic cells with constitutive lentiviral vectors are overcome by the use of transcriptionally targeted lentiviral vectors expressing WASp, which, at the same time, are efficient tools for gene therapy of WAS as demonstrated by their ability to reconstitute cellular defects from WASp-deficient mouse and human cells. We therefore postulate that transcriptionally regulated lentiviral vectors represent a safer and efficient alternative for the development of clinical protocols of WAS gene therapy.