RESUMO
Targeted gene transfer by nonviral vectors can be achieved through incorporation of specific ligand(s) into the vectors. In this study, the effects of incorporation of an anti-ErbB2 single-chain antibody fragment (ScFv) into nonviral vectors for targeted gene delivery were investigated. The ML39 ScFv, selected from a human ScFv phage display library and affinity matured in vitro (K(d)=1 x 10(-9) M), was used as ligand specific for the extracellular domain of the tumor surface protein, ErbB2. Two approaches were taken: (a) development of a vector that is composed of a bifunctional fusion protein capable of binding DNA with the ErbB2-specific ML39 ScFv at its N-terminus and a truncated form of human protamine at its C-terminus, and (b) formulation and evaluation of delivery vectors consisting of three independent components including ML39 ScFv, protamine, and cationic lipids. We demonstrate that fusion proteins comprised of the ML39 ScFv and a truncated form of protamine, denoted as ScFv-P-S, can selectively deliver exogenous DNA into ErbB2(+) cells, with an 8- to 10-fold increase in expression levels of the luciferase reporter gene in ErbB2(+) cells as compared to ErbB2(-) cells. In addition, vectors formulated by appropriately mixing DNA, ScFv, protamine, and lipids in vitro could even more efficiently deliver the reporter gene into ErbB2(+) cells with approximately 5-fold increase in gene expression in ErbB2(+) cell as compared to ErbB2(-) cells. Expression and refolding of the ScFv fusion proteins, in addition to determination of optimal conditions for vector development using these approaches, are discussed.
Assuntos
Neoplasias da Mama/terapia , Genes erbB-2/imunologia , Terapia Genética , Fragmentos de Imunoglobulinas/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Transfecção/métodos , Sequência de Aminoácidos , Fusão Gênica Artificial , Sítios de Ligação , Neoplasias da Mama/imunologia , Feminino , Citometria de Fluxo , Expressão Gênica , Genes erbB-2/genética , Vetores Genéticos , Humanos , Lipossomos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Protaminas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Homologia de Sequência de Aminoácidos , Transgenes , Células Tumorais CultivadasRESUMO
The mechanism(s) by which HIV-1 infection contributes to depletion of CD4(+) T cell is not well understood. In this report, we investigated whether a recently identified isoform of growth factor receptor bound protein (Grb2), named Grb3-3, a signaling molecule that is associated with the MAP kinase pathway and with apoptosis could be involved. We find that Grb3-3 is markedly up-regulated following HIV-1 infection of CD4(+) peripheral blood mononuclear cells undergoing apoptosis. Although IL-2 deprived CD4(+) cells also undergo apoptosis to a similar extent, Grb3-3 upregulation is not detected under these experimental conditions. Transient overexpression of Grb3-3 in Jurkat T-cells also causes apoptosis. Upon staurosporine stimulation, Grb3-3 predisposes Sup-T1 cell to apoptosis. Finally, analysis of the HIV-1 genes responsible for Grb3-3 expression demonstrates that Tat and Nef can independently induces its expression, suggesting these two earliest viral gene products of HIV-1 may share some common pathway(s) in up-regulating Grb3-3 expression.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Linfócitos T CD4-Positivos/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1 , Biossíntese de Proteínas , Apoptose/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Proteína Adaptadora GRB2 , Infecções por HIV/genética , Infecções por HIV/patologia , Humanos , Proteínas/genética , Regulação para CimaRESUMO
The MAPK pathway is required for T-cell activation; however, its role in modulating T-cell function following human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. In this report, we investigated whether Grb3-3, an isoform of the Grb2 (growth factor receptor-bound protein-2) adaptor molecule that is associated with the MAPK pathway, could be involved. We found that Grb3-3, but not its isoform Grb2, is markedly up-regulated in CD4(+) peripheral blood mononuclear cells derived from either in vitro HIV-1-infected cultures or HIV-1-infected human subjects. Analysis of HIV-1 gene products indicated that Tat and Nef, both of which have been implicated in modulating T-cell function, can independently induce expression of Grb3-3. By using NFAT/AP-1, AP-1, or NFAT reporter assays, we found that Grb3-3 can potentiate NFAT (but not AP-1) promoter activity in Jurkat T-cells upon engagement of the T-cell receptor and CD28 co-receptor. In addition, potentiation of NFAT by Grb3-3 is substantially suppressed by MEKK1, a kinase that may play an important role in retaining NFAT in the cytoplasm, and by cyclosporin A. Finally, we also found that Grb3-3 potentiates HIV-1 long terminal (LTR) repeat promoter activity following T-cell receptor stimulation, an effect that can be largely suppressed by cyclosporin A. Taken together, this study indicates that Grb3-3 is a cellular factor that can be up-regulated by HIV-1. In addition, Grb3-3 can also function as a positive factor for T-cell activation and, in doing so, may aid in establishing an intracellular environment that can optimally support HIV-1 replication.
