RESUMO
Immune responses against multiple epitopes are required for the prevention of hepatitis C virus (HCV) infection, and the progression to phase I trials of candidates may be guided by comparative immunogenicity studies in non-human primates. Four vectors, DNA, SFV, human serotype 5 adenovirus (HuAd5) and Modified Vaccinia Ankara (MVA) poxvirus, all expressing hepatitis C virus Core, E1, E2 and NS3, were combined in three prime-boost regimen, and their ability to elicit immune responses against HCV antigens in rhesus macaques was explored and compared. All combinations induced specific T-cell immune responses, including high IFN-γ production. The group immunized with the SFV+MVA regimen elicited higher E2-specific responses as compared with the two other modalities, while animals receiving HuAd5 injections elicited lower IL-4 responses as compared with those receiving MVA. The IFN-γ responses to NS3 were remarkably similar between groups. Only the adenovirus induced envelope-specific antibody responses, but these failed to show neutralizing activity. Therefore, the two novel regimens failed to induce superior responses as compared with already existing HCV vaccine candidates. Differences were found in response to envelope proteins, but the relevance of these remain uncertain given the surprisingly poor correlation with immunogenicity data in chimpanzees, underlining the difficulty to predict efficacy from immunology studies.
Assuntos
Linfócitos B/imunologia , Epitopos/genética , Hepacivirus/imunologia , Linfócitos T/imunologia , Vacinas contra Hepatite Viral/imunologia , Adenoviridae/genética , Animais , Linhagem Celular , Cricetinae , Epitopos/imunologia , Vetores Genéticos/genética , Imunogenicidade da Vacina , Interferon gama/sangue , Interleucina-4/sangue , Macaca mulatta , Masculino , Vaccinia virus/genética , Vacinas contra Hepatite Viral/genéticaRESUMO
Dendritic cells (DCs) are potent antigen-presenting cells capable of promoting or regulating innate and adaptive immune responses against non-self antigens. To better understand the DC biology or to use them for immune intervention, a tremendous effort has been made to improve gene transfer in these cells. Lentiviral vectors (LVs) have conferred a huge advantage in that they can transduce nondividing cells such as human monocyte-derived DCs (MDDCs) but required high amounts of viral particles and/or accessory proteins such as Vpx or Vpr to achieve sufficient transduction rates. As a consequence, these LVs have been shown to cause dramatic functional modifications, such as the activation or maturation of transduced MDDCs. Taking advantage of new pseudotyped LVs, i.e., with envelope glycoproteins from the measles virus (MV), we demonstrate that MDDCs are transduced very efficiently with these new LVs compared to the classically used vesicular stomatitis virus G-pseudotyped LVs and thus allowed to achieve high transduction rates at relatively low multiplicities of infection. Moreover, in this experimental setting, no activation or maturation markers were upregulated, while MV-LV-transduced cells remained able to mature after an appropriate Toll-like receptor stimulation. We then demonstrate that our MV-pseudotyped LVs use DC-SIGN, CD46, and CD150/SLAM as receptors to transduce MDDCs. Altogether, our results show that MV-pseudotyped LVs provide the most accurate and simple viral method for efficiently transferring genes into MDDCs without affecting their activation and/or maturation status.
Assuntos
Células Dendríticas/metabolismo , Vetores Genéticos/genética , Lentivirus/genética , Vírus do Sarampo/genética , Transdução Genética , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Envelope Viral/genética , Moléculas de Adesão Celular/genética , Diferenciação Celular , Células Dendríticas/citologia , Células Dendríticas/virologia , Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Lectinas Tipo C/genética , Proteína Cofatora de Membrana/genética , Receptores de Superfície Celular/genética , Fase de Repouso do Ciclo Celular , Receptor 3 Toll-Like/agonistasRESUMO
Transgenic rabbit is the preferred disease model of atherosclerosis, lipoprotein metabolism and cardiovascular diseases since upon introducing genetic mutations of human genes, rabbit models reflect human physiological and pathological states more accurately than mouse models. Beyond that, transgenic rabbits are also used as bioreactors to produce pharmaceutical proteins in their milk. Since in the laboratory rabbit the conventional transgenesis has worked with the same low efficiency in the last twenty five years and truly pluripotent embryonic stem cells are not available to perform targeted mutagenesis, our aim was to adapt lentiviral transgenesis to this species. A simian immunodeficiency virus based replication defective lentiviral vector was used to create transgenic rabbit through perivitelline space injection of fertilized oocytes. The enhanced green fluorescent protein (GFP) gene was placed under the ubiquitous CAG promoter. Transgenic founder rabbits showed mosaic pattern of GFP expression. Transgene integration and expression was revealed in tissues derived from all three primary germ layers. Transgene expression was detected in the developing sperm cells and could get through the germ line without epigenetic silencing, albeit with very low frequency. Our data show for the first time, that lentiviral transgenesis could be a feasible and viable alternative method to create genetically modified laboratory rabbit.
Assuntos
Animais Geneticamente Modificados/genética , Vetores Genéticos/genética , Coelhos/genética , Vírus da Imunodeficiência Símia/genética , Transgenes , Animais , Transferência Embrionária , Estudos de Viabilidade , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Masculino , Microinjeções , Mosaicismo , Especificidade de Órgãos , Espermatozoides/química , ZigotoRESUMO
Wiskott-Aldrich syndrome (WAS) gene therapy requires highly efficient and well-controlled vectors. Here we studied the performance of a lentiviral vector (LV) harbouring a 500-bp fragment of the WAS proximal promoter (WW), which we previously characterized as haematopoietic-specific and capable of restoring WAS phenotype in patients' T cells. We used an LV (WE) expressing eGFP to evaluate whether this promoter was following the expression pattern of endogenous WASp. Transgene expression was analysed in WE-transduced hCD34+ population and its progeny after in vitro and in vivo differentiation in the Rag2-/-, gammac-/- humanized mouse. We revealed very poor expression from the WE internal promoter in macrophages and erythroid cells. Therefore, we designed a novel LV including a fragment of the alternative WAS promoter in WE vector (AWE). This new vector sustained high transgene levels along the whole lymphoid lineage in vivo. Most importantly, the performance of AWE vector was highly superior to WE vector since AWE clearly improved transgene levels in in vitro and in vivo hCD34+-derived macrophages, erythroid cells, megakaryocytes and B cells while supporting a high expression in human T cells. This emphasizes that it is a suitable LV backbone for gene therapy of haematopoietic diseases such as WAS.
Assuntos
Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Lentivirus/genética , Regiões Promotoras Genéticas , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Animais , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Expressão Gênica , Engenharia Genética , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Hematopoese , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transdução Genética/métodos , Transgenes , Proteína da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
A major limitation of current lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent cells, such as human CD34+ cells that reside in the G0 phase of the cell cycle and that are highly enriched in hematopoietic stem cells. This hampers their application for gene therapy of hematopoietic cells. We describe here novel LVs that overcome this restriction by displaying early-acting cytokines on their surface. Display of thrombopoietin, stem cell factor or both cytokines on LV surface allows high transfer into quiescent cord blood CD34+ cells. Moreover, these surface-engineered LVs preferentially transduce and promote survival of resting CD34+ cells rather than cycling cells. These novel LVs allow superior gene transfer in the most immature CD34+ cells compared to conventional LVs, even in the presence of recombinant cytokines. This is demonstrated by their capacity to promote selective transduction in long-term culture initiating cell colonies (LTC-ICs) and of long-term non-obese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRCs). Here we describe the production of these "early acting cytokine" displaying vectors and the methodology to confirm the capacity of these vectors to promote selective transduction of HSCs.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Células-Tronco Hematopoéticas/fisiologia , Lentivirus/fisiologia , Fator de Células-Tronco/metabolismo , Trombopoetina/metabolismo , Animais , Antígenos CD34/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transdução GenéticaRESUMO
Lentiviral vectors are efficiently pseudotyped with RD114-TR, a chimeric envelope glycoprotein made of the extracellular and transmembrane domains of the feline leukemia virus RD114 and the cytoplasmic tail of the murine leukemia virus amphotropic envelope. RD114-TR-pseudotyped vectors may be concentrated by centrifugation, are resistant to complement inactivation, and are suitable for both ex vivo and in vivo gene therapy applications. We analyzed RD114-TR-pseudotyped, HIV-1-derived lentiviral vectors for their ability to transduce human cord blood, bone marrow, and peripheral blood mobilized CD34(+) hematopoietic stem/progenitor cells. Transduction efficiency was analyzed in CD34(+) cells in liquid culture, in CD34(+) clonogenic progenitors in semisolid culture, and in CD34(+) repopulating stem cells after xenotransplantation in NOD-SCID mice. Compared with a standard VSV-G-based packaging system, RD114-TR-pseudotyped particles transduced hematopoietic stem/progenitor cells at lower multiplicity of infection, with lower toxicity and less pseudo-transduction at comparable vector copy number per genome. Potential changes in the CD34(+) cell transcription profile and phenotype on transduction with RD114-TR-pseudotyped vectors was comparatively investigated by microarray analysis. Our study shows that the biology of repopulating hematopoietic stem cells and their progeny is not affected by transduction with RD114-TR-pseudotyped lentiviral vectors. RD114-TR is compatible with the development of lentiviral stable packaging cell lines, and may become the envelope of choice for clinical studies aiming at safe and efficient genetic modification of human hematopoietic stem cells.
Assuntos
Vetores Genéticos , Células-Tronco Hematopoéticas/fisiologia , Lentivirus/genética , Transdução de Sinais , Proteínas do Envelope Viral/genética , Animais , Antígenos CD34/sangue , Antígenos CD34/imunologia , Antígenos CD34/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Vírus da Imunodeficiência Felina/química , Vírus da Leucemia Murina/genética , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Transplante HeterólogoRESUMO
IGF-I regulates pituitary and gonadal functions, and is pivotal for sexual development and fertility in mammalian species. To better understand the function of autocrine IGF-I in Sertoli cell physiology, we established a system for Cre-mediated conditional inactivation of the IGF-I receptor (IGF-IR) in cultured Sertoli cells. We show here that loss of IGF-IR decreased the number of viable Sertoli cells as a consequence of diminished Sertoli cell proliferation and increased Sertoli cell death. Furthermore, the lack of IGF-IR altered the morphology of cultured Sertoli cells and decreased lactate and transferrin secretions. Collectively, our data indicate that autocrine IGF-I contributes significantly to Sertoli cell homeostasis. The described in vitro system for loss-of-function analysis of the IGF-IR can be readily transposed to study the role of other intratesticular growth factors involved in spermatogenesis.
Assuntos
Comunicação Autócrina/fisiologia , Fator de Crescimento Insulin-Like I/metabolismo , Receptor IGF Tipo 1/genética , Células de Sertoli/metabolismo , Espermatogênese/fisiologia , Animais , Proliferação de Células , Sobrevivência Celular , Engenharia Genética , Immunoblotting , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Phage display libraries are widely used for selection and optimization of polypeptide ligands or protease substrates. Because they are expressed and amplified in bacterial hosts, phage are not ideal for displaying eukaryotic polypeptides or for probing mammalian cells. As retroviruses do not suffer from these limitations we constructed plasmids encoding replication-competent murine leukemia viruses displaying a virally encoded epidermal growth factor (EGF) domain at the N-terminus of the envelope glycoprotein. The EGF-displaying viruses replicated freely on EGF receptor-poor cells without deleting the displayed EGF domain but did not propagate on EGF receptor-rich cells because they were sequestered by the EGF receptors. A retrovirus display library was then generated by diversifying the seven-residue linker between the envelope glycoprotein and the displayed EGF domain. Selective pressure for loss of EGF receptor-binding activity was applied to the library by serial passage on EGF receptor-rich HT1080 cells. The selected viruses propagated on these cells with wild-type efficiencies, a phenotype that was conferred by intracellular cleavage of their displayed linker sequences. The selected linker sequences invariably presented arginine-rich motifs matching the consensus cleavage signal for furin-like proteases. Retrovirus display libraries can be used for the selection of polypeptides interacting with components of living mammalian cells.
Assuntos
Endopeptidases/metabolismo , Vírus da Leucemia Murina/genética , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Hidrólise , Camundongos , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
We report here the use of viral fusogenic membrane glycoproteins (FMGs) as a new class of therapeutic genes for the control of tumor growth. FMGs kill cells by fusing them into large multinucleated syncytia, which die by sequestration of cell nuclei and subsequent nuclear fusion by a mechanism that is nonapoptotic, as assessed by multiple criteria. Direct and bystander killing of three different FMGs were at least one log more potent than that of herpes simplex virus thymidine kinase or cytosine deaminase suicide genes. Transduction of human tumor xenografts with plasmid DNA prevented tumor outgrowth in vivo, and cytotoxicity could be regulated through transcriptional targeting. Syncytial formation is accompanied by the induction of immunostimulatory heat shock proteins, and tumor-associated FMG expression in immunocompetent animals generated specific antitumor immunity.
Assuntos
Fusão Celular/genética , Terapia Genética/métodos , Neoplasias Experimentais/terapia , Proteínas Virais de Fusão/genética , Animais , Apoptose/genética , Divisão Celular/genética , Fusão Celular/imunologia , Linhagem Celular , DNA Complementar/genética , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Plasmídeos/genética , Simplexvirus/enzimologia , Timidina Quinase/genética , Transfecção , Células Tumorais Cultivadas , Proteínas Virais de Fusão/imunologiaRESUMO
Hepatitis C virus (HCV), an important human pathogen, is an enveloped, positive-stranded RNA virus classified in the hepacivirus genus of the Flaviviridae family. Cell attachment of flaviviruses generally leads to endocytosis of bound virions. Systems that support HCV replication and particle formation in vitro are emerging only now, 16 years after the discovery of the virus. Albeit this limitation, the route of HCV cell entry as well as 'capture' molecules involved in low-affinity interactions for the initial contact of HCV with target cells and potential high-affinity receptor candidates that may mediate HCV trafficking and fusion have been described. The objective of this review is to summarise the contribution of different HCV model systems to our current knowldege about structure of theHCVGPs E1 and E2 and their roles in cell entry comprising cell attachment, interactions with cellular receptors, endocytosis and fusion.
RESUMO
Essentials B cells are attractive targets for gene therapy and particularly interesting for immunotherapy. A baboon envelope pseudotyped lentiviral vector (BaEV-LV) was tested for B-cell transduction. BaEV-LVs transduced mature and plasma human B cells with very high efficacy. BaEV-LVs allowed secretion of functional factor IX from B cells at therapeutic levels in vivo. SUMMARY: Background B cells are attractive targets for gene therapy for diseases associated with B-cell dysfunction and particularly interesting for immunotherapy. Moreover, B cells are potent protein-secreting cells and can be tolerogenic antigen-presenting cells. Objective Evaluation of human B cells for secretion of clotting factors such as factor IX (FIX) as a possible treatment for hemophilia. Methods We tested here for the first time our newly developed baboon envelope (BaEV) pseudotyped lentiviral vectors (LVs) for human (h) B-cell transduction following their adaptive transfer into an NOD/SCIDγc-/- (NSG) mouse. Results Upon B-cell receptor stimulation, BaEV-LVs transduced up to 80% of hB cells, whereas vesicular stomatitis virus G protein VSV-G-LV only reached 5%. Remarkably, BaEVTR-LVs permitted efficient transduction of 20% of resting naive and 40% of resting memory B cells. Importantly, BaEV-LVs reached up to 100% transduction of human plasmocytes ex vivo. Adoptive transfer of BaEV-LV-transduced mature B cells into NOD/SCID/γc-/- (NSG) [non-obese diabetic (NOD), severe combined immuno-deficiency (SCID)] mice allowed differentiation into plasmablasts and plasma B cells, confirming a sustained high-level gene marking in vivo. As proof of principle, we assessed BaEV-LV for transfer of human factor IX (hFIX) into B cells. BaEV-LVs encoding FIX efficiently transduced hB cells and their transfer into NSG mice demonstrated for the first time secretion of functional hFIX from hB cells at therapeutic levels in vivo. Conclusions The BaEV-LVs might represent a valuable tool for therapeutic protein secretion from autologous B cells in vivo in the treatment of hemophilia and other acquired or inherited diseases.
Assuntos
Linfócitos B/citologia , Fator IX/metabolismo , Vetores Genéticos , Hemofilia A/sangue , Lentivirus/genética , Animais , Células Apresentadoras de Antígenos/citologia , Linfócitos T CD4-Positivos/citologia , Proliferação de Células , Técnicas de Transferência de Genes , Células HEK293 , Hemofilia A/terapia , Humanos , Imunoterapia/métodos , Glicoproteínas de Membrana/química , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Papio , Plasmídeos , Transdução Genética , Transgenes , Resultado do Tratamento , Proteínas do Envelope Viral/químicaRESUMO
The avian myeloblastosis virus (AMV) causes monoblastic leukemia in the chick. Two non-producer clones of AMV-transformed monoblasts, BM2/C3A and BM2L/A2B5, have been described (see Bottazzi et al., this issue). They differ in their growth requirements and in their ability to induce leukemia when injected into the chick embryo. We first genetically tagged these clones by retroviral infection with a vector expressing the bacterial lacZ gene. Then, we injected the lacZ-positive cells via the chorioallantoic vein into chick embryos. With BM2L/A2B5 cells, the bone marrow of the injected birds was rapidly invaded by lacZ-positive cells. In addition, these cells rapidly overgrew cultures of bone marrow cells derived from injected animals. Conversely, the growth of BM2/C3A was inhibited in the injected animals and only a few blue cells, with the morphology of macrophages, were detected in cultures of bone marrow cells. We developed an in vitro assay to mimic in vitro the differential growth of BM2/C3A and BM2L/A2B5 observed in vivo. These data strongly suggest that BM2/C3A cells retain their ability to differentiate into macrophages in the normal bone marrow environment and that BM2L/A2B5 cells differ from BMC/C3A in the loss of this capacity.
Assuntos
Medula Óssea/fisiologia , Transformação Celular Neoplásica , Leucemia Monocítica Aguda/genética , Oncogenes , Proteínas Oncogênicas de Retroviridae/genética , Animais , Comunicação Celular , Divisão Celular , Linhagem Celular Transformada , Embrião de Galinha , Óperon Lac , Leucemia Monocítica Aguda/etiologia , Proteínas Oncogênicas v-mybRESUMO
We have displayed insulin-like growth factor I (IGF-I) as an N-terminal extension of 4070A (amphotropic) retroviral envelope protein. Western blot demonstrated that chimaeric envelope proteins were incorporated into retroviral particles. Interaction between the displayed IGF-I and cell-surface receptors impaired gene delivery. The magnitude of this inhibitory effect was smallest on NIH 3T3 cells, greater on NIH 3T3 cells over-expressing insulin receptor, and greatest on NIH 3T3 cells over-expressing human type-I IGF receptor. Hence, both the number of ligand receptors and their affinity for the displayed ligand influenced the level of gene delivery. The inhibitory effect was abrogated by cleaving the displayed domain from the underlying envelope protein with factor Xa protease, and by the addition of free ligand to the infection. Addition of IGF-I or insulin caused a dose-dependent increase in titre. Possible mechanisms for receptor-mediated inhibition of gene delivery by IGF-displaying vectors are discussed, together with the implications of these results for practical applications of retroviral display and for understanding the mechanism of virus entry.
Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Retroviridae/fisiologia , Células 3T3 , Animais , Fator de Crescimento Epidérmico/biossíntese , Fator de Crescimento Epidérmico/genética , Receptores ErbB/metabolismo , Vetores Genéticos/fisiologia , Humanos , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/genética , Camundongos , Receptor IGF Tipo 1/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tropismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vírion/fisiologia , Montagem de VírusRESUMO
Retroviral vectors capable of efficient in vivo gene delivery to specific target cell types or to specific locations of disease pathology would greatly facilitate many gene therapy applications. The surface glycoproteins of membrane-enveloped viruses stand among the choice candidates to control the target cell receptor recognition and host range of retroviral vectors onto which they are incorporated. This can be achieved in many ways, such as the exchange of glycoprotein by pseudotyping, their biochemical modifications, their conjugation with virus-cell bridging agents or their structural modifications. Understanding the fundamental properties of the viral glycoproteins and the molecular mechanism of virus entry into cells has been instrumental in the functional alteration of their tropism. Here we briefly review the current state of our understanding of the structure and function of viral envelope glycoproteins and we discuss the emerging targeting strategies based on retroviral and lentiviral vector systems.
Assuntos
Vetores Genéticos/genética , Glicoproteínas/metabolismo , Lentivirus/genética , Retroviridae/genética , Marcação de Genes , Técnicas de Transferência de Genes , Humanos , Receptores de Superfície Celular/metabolismo , Retroviridae/fisiologiaRESUMO
Retroviral vectors with the capacity to deliver transgenes to specific tissues are expected to be of great value for various gene transfer applications in vivo. Initial attempts to modify vector host-range by the insertion of ligands on their surface glycoproteins have frequently failed, essentially owing to the impairment of the fusogenicity of the vector particles bound to the targeted cell-surface molecules. Several strategies aimed to recover the fusogenic activity of surface-engineered vector particles have recently been explored and have given rise to novel concepts in the field.
Assuntos
Marcação de Genes/métodos , Técnicas de Transferência de Genes , Vetores Genéticos , Retroviridae/genética , Proteínas Virais de Fusão/genética , Animais , Fusão de Membrana , Modelos Biológicos , Engenharia de Proteínas/métodos , Receptores Virais/metabolismo , Especificidade da Espécie , Proteínas Virais de Fusão/fisiologia , Vírion/genética , Vírion/metabolismoRESUMO
Defective avian leukosis-based vectors expressing the bacterial lacZ gene were used as helper-free preparations to infect early stage Brown-Leghorn embryos. Both in toto X-gal staining and DNA analysis using Southern blot technique were applied to detect virus integration and expression. Our results demonstrate a low efficiency of in vitro infection in early stages of embryonic development. Southern blot analysis reveals that only 1% of embryonic cells integrate the vector genome after infection using 2 to 12 virus particle per embryonic cell. In situ expression of the lacZ marker gene was detected in only 0.06% of embryonic cells. These results lead us to conclude that only 6% of infected cells express efficiently the lacZ marker gene. This low level of expression could result from avian leukosis virus LTRs inhibition in chicken embryonic cells at an early stage of development. In spite of the low efficiency of infection, no evidence for tissue restrictive expression was observed. However, vector containing LTRs from RAV-2 virus allows preferential expression of provirus vector in neural tube tissue, whereas cardiac localization of the preferential expression was observed using vector containing the RAV-1 LTRs. The chronological analysis of the marker gene expression in terms of location of expression foci and sizes of these foci, lead us to hypothesize the putative regulation of retrovirus expression linked to embryonic development.
Assuntos
Vírus da Leucose Aviária/genética , Regulação Viral da Expressão Gênica , Óperon Lac , Retroviridae/genética , Animais , Animais Geneticamente Modificados , Southern Blotting , Embrião de Galinha , Vetores Genéticos , Microinjeções , beta-Galactosidase/análiseRESUMO
We have used high-titer (10(8) ffu/ml) recombinant retroviral vectors to transfer the beta-galactosidase (beta-Gal) gene to rat hepatocytes in vivo. In animals injected twice in the portal blood stream the next day after partial hepatectomy, half of the hepatocytes (46 +/- 17%) expressed the marker at the end of liver regeneration. The number of positive cells closely correlated with the viral titer as well as with beta-Gal enzymatic activity present in the whole liver. Because genes transferred via retroviral vectors in the liver are known to be expressed permanently, our present results open new possibilities for the development of gene therapy protocols for hereditary liver diseases using recombinant retroviral vectors.
Assuntos
Técnicas de Transferência de Genes , Fígado/virologia , Retroviridae/genética , Animais , Vetores Genéticos/farmacologia , Hepatectomia , Fígado/metabolismo , Masculino , Ratos , Ratos Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução Genética , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
We have generated three different E1-deleted replication-defective adenoviral vectors expressing either Moloney murine leukemia virus (Mo-MuLV) Gag-Pol core particle proteins, gibbon ape leukemia virus (GALV) envelope glycoproteins, or an MuLV-derived retroviral vector genome encoding mCD2 antigen, a murine cell surface marker easily detectable by flow cytometry. Each of the three vectors was first characterized individually by infection of cells providing the complementary retroviral function(s) and able to induce the production of retroviral vectors with an efficiency similar to or higher than that of FLY stable retroviral packaging cells [Cosset, F.-L., Takeuchi, Y., Battini, J.-L., Weiss, R.A., and Collins, M.K.L., (1995). J. Virol. 69, 7430-7436]. In small-scale pilot experiments, TE671 cells simultaneously coinfected with the three adenoviral vectors efficiently released helper-free retroviral vectors in their supernatant, with titers greater than 10(6) infectious particles per milliliter by end-point titrations. Our results also indicated that in contrast to retroviral vector-packageable RNAs, the adenovirus-mediated overexpression of both Gag-Pol and Env packaging functions had limited impact on retroviral titers. The primary mechanism suspected is the premature intracellular cleavage of the Pr65gag precursor that we found in gag-pol-expressing cells, which in turn may impair the normal incorporation of high loads of functional Env. Last, the characterization of the adenoviral/retroviral chimeric vectors allowed the screening of various primate cells for retroviral production and we found that three hepatocyte-derived cell lines were highly efficient in the assembly and release of infectious retroviral particles.
Assuntos
Adenoviridae/genética , Vetores Genéticos , Retroviridae/genética , Animais , Antígenos CD2/genética , Linhagem Celular , Quimera/genética , Humanos , Recombinação Genética , Proteínas Virais/genética , Montagem de VírusRESUMO
We are developing protease-activatable gene delivery vehicles for selective gene delivery to protease-expressing cells. Angiogenesis, inflammation, and cancer invasion are linked to the overexpression of matrix metalloproteinases (MMPs), which destroy the extracellular matrix. Therefore, the MMPs are promising targets for therapy. We have displayed epidermal growth factor (EGF) on retroviral vector particles as an MMP-cleavable amino-terminal extension of the 4070A murine leukemia virus (MLV) envelope glycoprotein. This was achieved by engineering an MMP-cleavage signal (PLGLWA) into the linker between the EGF domain and the 4070A SU. The chimeric envelope was expressed and incorporated into viral particles, and the EGF domain could be cleaved from the surface of the viral particles by gelatinase A (MMP-2). The MMP-sensitive vector and control MMP-insensitive vectors could bind, via their displayed EGF domains, to EGF receptors on A431 cells but were unable to infect them because the EGF receptor (EGFR) does not support postbinding steps required for retroviral entry. In the presence of exogenous MMPs, the infectivity of the MMP-sensitive vector, but not of the MMP-insensitive vectors, was restored on A431 cells, and this cleavage activation could be partially blocked by MMP inhibitors. Endogenous MMPs produced by EGFR-positive HT 1080 cells could selectively activate the MMP-sensitive vector giving rise to a titer that was 1,000-fold higher on HT 1080 cells than on MMP-negative A431 cells. Inhibitor studies and gelatin zymograms indicated that the membrane-associated MT-MMP expressed on the HT 1080 cells played an important role in cleavage activation of the vector. When presented simultaneously with both EGFR-positive cell lines A431 and HT 1080, the vector could efficiently discriminate between the two different cell types, infecting the MMP-positive HT 1080 cells in preference over the A431 cells.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Metaloendopeptidases/metabolismo , Retroviridae , Sítios de Ligação , Receptores ErbB/metabolismo , Fator Xa/metabolismo , Gelatinases/metabolismo , Engenharia Genética , Glicoproteínas/metabolismo , Humanos , Metaloproteinase 2 da Matriz , Vírus da Leucemia Murina de Moloney , Inibidores de Proteases/metabolismo , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Análise de Sequência de DNA , Inibidor Tecidual de Metaloproteinase-2 , Inibidores Teciduais de Metaloproteinases , Células Tumorais Cultivadas , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Dendritic cells (DCs), the most potent antigen-presenting cells, can be generated from CD34+ hematopoietic stem cells and used for generating therapeutic immune responses. To develop immunotherapy protocols based on genetically modified DCs, we have investigated the conditions for high-level transduction of a large amount of CD34+-derived DCs. Thus, we have used an efficient and clinically applicable protocol for the retroviral transduction of cord blood (CB) or mobilized peripheral blood (MPB) CD34+ cells based on infection with gibbon ape leukemia virus (GALV)-pseudotyped retroviral vectors carrying the nls-LacZ reporter gene. Infected cells have been subsequently cultured under conditions allowing their dendritic differentiation. The results show that using a growth factor combination including granulocyte-macrophage colony-stimulating factor plus tumor necrosis factor alpha plus interleukin 4 plus stem cell factor plus Flt3 ligand, more than 70% of DCs derived from CB or MPB CD34+ cells can be transduced. Semiquantitative PCR indicates that at least two proviral copies per cell were detected. Transduced DCs retain normal immunophenotype and potent T cell stimulatory capacity. Finally, by using a semisolid methylcellulose assay for dendritic progenitors (CFU-DCs), we show that more than 90% of CFU-DCs can be transduced. Such a highly efficient retrovirus-mediated gene transfer into CD34+-derived DCs makes it possible to envision the use of this methodology in clinical trials.