RESUMO
As sentinels of the immune system, dendritic cells (DCs) play an essential role in regulating cellular immune responses. One of the main challenges of developing DC-targeted therapies includes the delivery of antigen to DCs in order to promote the activation of antigen-specific effector CD8 T cells. With the goal of creating antigen-directed immunotherapeutics that can be safely administered directly to patients, Immune Design has developed a platform of novel integration-deficient lentiviral vectors that target and deliver antigen-encoding nucleic acids to human DCs. This platform, termed ID-VP02, utilizes a novel genetic variant of a Sindbis virus envelope glycoprotein with posttranslational carbohydrate modifications in combination with Vpx, a SIVmac viral accessory protein, to achieve efficient targeting and transduction of human DCs. In addition, ID-VP02 incorporates safety features in its design that include two redundant mechanisms to render ID-VP02 integration-deficient. Here, we describe the characteristics that allow ID-VP02 to specifically transduce human DCs, and the advances that ID-VP02 brings to conventional third-generation lentiviral vector design as well as demonstrate upstream production yields that will enable manufacturing feasibility studies to be conducted.
Assuntos
Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Lentivirus/genética , Sindbis virus/genética , Proteínas do Envelope Viral/genética , Vetores Genéticos/administração & dosagem , Células HEK293 , Humanos , Imunidade Celular/imunologia , Distribuição TecidualRESUMO
Peripheral CD4(+)Vß5(+) T cells are tolerized to an endogenous mouse mammary tumor virus superantigen either by deletion or TCR revision. Through TCR revision, RAG reexpression mediates extrathymic TCRß rearrangement and results in a population of postrevision CD4(+)Vß5(-) T cells expressing revised TCRß chains. We have hypothesized that cell death pathways regulate the selection of cells undergoing TCR revision to ensure the safety and utility of the postrevision population. In this study, we investigate the role of Bcl-2-interacting mediator of cell death (Bim)-mediated cell death in autoantigen-driven deletion and TCR revision. Bim deficiency and Bcl-2 overexpression in Vß5 transgenic (Tg) mice both impair peripheral deletion. Vß5 Tg Bim-deficient and Bcl-2 Tg mice exhibit an elevated frequency of CD4(+) T cells expressing both the transgene-encoded Vß5 chain and a revised TCRß chain. We now show that these dual-TCR-expressing cells are TCR revision intermediates and that the population of RAG-expressing, revising CD4(+) T cells is increased in Bim-deficient Vß5 Tg mice. These findings support a role for Bim and Bcl-2 in regulating the balance of survival versus apoptosis in peripheral T cells undergoing RAG-dependent TCR rearrangements during TCR revision, thereby ensuring the utility of the postrevision repertoire.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Autoantígenos/fisiologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Depleção Linfocítica , Proteínas de Membrana/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Antígenos de Linfócitos T/biossíntese , Animais , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/deficiência , Proteína 11 Semelhante a Bcl-2 , Linfócitos T CD4-Positivos/virologia , Morte Celular/genética , Morte Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Rearranjo Gênico do Linfócito T/imunologia , Genes RAG-1/imunologia , Humanos , Vírus do Tumor Mamário do Camundongo/imunologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/deficiência , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Hox genes control many developmental events along the AP axis, but few target genes have been identified. Whether target genes are activated or repressed, what enhancer elements are required for regulation, and how different domains of the Hox proteins contribute to regulatory specificity are poorly understood. Six2 is genetically downstream of both the Hox11 paralogous genes in the developing mammalian kidney and Hoxa2 in branchial arch and facial mesenchyme. Loss-of-function of Hox11 leads to loss of Six2 expression and loss-of-function of Hoxa2 leads to expanded Six2 expression. Herein we demonstrate that a single enhancer site upstream of the Six2 coding sequence is responsible for both activation by Hox11 proteins in the kidney and repression by Hoxa2 in the branchial arch and facial mesenchyme in vivo. DNA-binding activity is required for both activation and repression, but differential activity is not controlled by differences in the homeodomains. Rather, protein domains N- and C-terminal to the homeodomain confer activation versus repression activity. These data support a model in which the DNA-binding specificity of Hox proteins in vivo may be similar, consistent with accumulated in vitro data, and that unique functions result mainly from differential interactions mediated by non-homeodomain regions of Hox proteins.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Isoformas de Proteínas/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Região Branquial/anatomia & histologia , Região Branquial/embriologia , Região Branquial/metabolismo , DNA/metabolismo , Genes Reporter , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Isoformas de Proteínas/genética , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genéticaRESUMO
Current laboratory methods for comprehensive thrombophilia status require large blood volumes and long turn-around times. We demonstrate the feasibility of performing thrombophilia panel testing of enzymatic functional assays on a microfluidic cartridge using low sample volume.Functional assays for Antithrombin III, Protein C, Factor VIII, and plasminogen were adapted on the digital microfluidic platform by developing novel fluorogenic substrates and establishing on-cartridge fluorescence (360/460 nm) detection. Cartridge vs. microtiter plate results were compared using samples obtained from pediatric patients. Linear regression and Bland-Altman plots were used to establish correlations. Results were not significantly different when performed on-cartridge compared to microtiter plates. Importantly, the sample volume required is significantly lower for all on-cartridge compared to microtiter plate assays (25 µL vs. 2 ml).This study demonstrates the feasibility of thrombophilia panel testing with high-fidelity using small plasma volume. The efficacy of this near-patient technology in clinical settings needs further investigation.
Assuntos
Transtornos da Coagulação Sanguínea/diagnóstico , Testes de Coagulação Sanguínea/métodos , Ensaios Enzimáticos/métodos , Técnicas Analíticas Microfluídicas/métodos , Testes de Coagulação Sanguínea/normas , Humanos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos TestesRESUMO
Dendritic cells (DCs) are essential antigen-presenting cells for the initiation of cytotoxic T-cell responses and therefore attractive targets for cancer immunotherapy. We have developed an integration-deficient lentiviral vector termed ID-VP02 that is designed to deliver antigen-encoding nucleic acids selectively to human DCs in vivo. ID-VP02 utilizes a genetically and glycobiologically engineered Sindbis virus glycoprotein to target human DCs through the C-type lectin DC-SIGN (CD209) and also binds to the homologue murine receptor SIGNR1. Specificity of ID-VP02 for antigen-presenting cells in the mouse was confirmed through biodistribution studies showing that following subcutaneous administration, transgene expression was only detectable at the injection site and the draining lymph node. A single immunization with ID-VP02 induced a high level of antigen-specific, polyfunctional effector and memory CD8 T-cell responses that fully protected against vaccinia virus challenge. Upon homologous readministration, ID-VP02 induced a level of high-quality secondary effector and memory cells characterized by stable polyfunctionality and expression of IL-7Rα. Importantly, a single injection of ID-VP02 also induced robust cytotoxic responses against an endogenous rejection antigen of CT26 colon carcinoma cells and conferred both prophylactic and therapeutic antitumor efficacy. ID-VP02 is the first lentiviral vector which combines integration deficiency with DC targeting and is currently being investigated in a phase I trial in cancer patients.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer , Carcinoma/terapia , Neoplasias do Colo/terapia , Células Dendríticas/imunologia , Vetores Genéticos , Imunoterapia Adotiva , Lentivirus/genética , Sindbis virus/genética , Vaccinia virus/imunologia , Vacínia/imunologia , Animais , Carcinoma/imunologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Ensaios Clínicos Fase I como Assunto , Neoplasias do Colo/imunologia , Citotoxicidade Imunológica , Células Dendríticas/transplante , Células Dendríticas/virologia , Engenharia Genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Memória Imunológica , Lectinas Tipo C/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ligação Proteica , Receptores de Superfície Celular/metabolismo , Receptores de Interleucina-7/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Integração Viral/genéticaRESUMO
Hox genes are crucial for body axis specification during embryonic development. Hoxa11 plays a role in anteroposterior patterning of the axial skeleton, development of the urogenital tract of both sexes, and proximodistal patterning of the limbs. Hoxa11 expression is also observed in the neural tube. Herein, we report the generation of a Hoxa11eGFP targeted knock-in allele in mice in which eGFP replaces the first coding exon of Hoxa11 as an in-frame fusion. This allele closely recapitulates the reported mRNA expression patterns for Hoxa11. Hoxa11eGFP can be visualized in the tail, neural tube, limbs, kidneys, and reproductive tract of both sexes. Additionally, homozygous mutants recapitulate reported phenotypes for Hoxa11 loss of function mice, exhibiting loss of fertility in both males and females. This targeted mouse line will prove useful as a vital marker for Hoxa11 protein localization during control (heterozygous) or mutant organogenesis.