RESUMEN
Condensing peptide-DNA complexes have great potential as nonviral agents for gene delivery. To date, however, such complexes have given transfection activities greatly inferior to adenovirus and somewhat inferior to cationic lipid-DNA complexes, even for cell lines and primary cells in vitro. We report here the identification of a novel condensing peptide, CL22, which forms DNA complexes that efficiently transfect many cell lines, as well as primary dendritic and endothelial cells. We report studies with sequence and structure variants that define some properties of the peptide that contribute to efficient transfection. We demonstrate that the superior transfection activity of CL22 compared with other DNA condensing peptides is conferred at a step after uptake of the complexes into cells. We show that CL22-DNA complexes have transfection activity that is at least equivalent to the best available nonviral agents.
Asunto(s)
Fragmentos de Péptidos/genética , Péptidos/genética , Transfección/métodos , Secuencia de Aminoácidos , Animales , Técnicas de Cultivo de Célula , ADN/genética , Células Dendríticas/metabolismo , Endotelio Vascular/citología , Vectores Genéticos , Humanos , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Péptidos/química , Células Tumorales CultivadasRESUMEN
Currently, the limited supply and stability of some human autoantigens pose formidable difficulties in characterizing patients' T cells specific for them; recombinant preparations may contain bacterial contaminants, and synthetic peptides have arbitrarily chosen start and stop points. In order to provide a stable antigen source with naturally processed epitopes, a full-length acetylcholine receptor (AChR) alpha subunit construct was transfected into B-lymphoblastoid cell lines (B-LCL). Expression was much easier to detect at the mRNA level than the protein level. Nevertheless, this transfectant also stimulated a T-cell line that recognized the alpha 149-156 region in the context of HLA-DR4 at high sensitivity. The responses were specific both for the antigen transfected and for the presenting HLA-DR allele. This study thus confirms the potential of autologous B-LCL expressing natural epitopes in the context of HLA class II molecules for characterizing established T-cell lines, and perhaps also for initiating new ones.
Asunto(s)
Autoantígenos/genética , Linfocitos B , Linfocitos T CD4-Positivos/inmunología , Receptores Colinérgicos/inmunología , Transfección , Linfocitos B/inmunología , Northern Blotting , Western Blotting , Línea Celular , Humanos , Yoduro Peroxidasa/genética , Yoduro Peroxidasa/metabolismo , ARN Mensajero/metabolismo , Receptores Colinérgicos/genéticaRESUMEN
The TSH-receptor (TSH-R) and thyroid peroxidase (TPO) are targets of autoantibody production in the autoimmune thyroid disease, Graves' disease, and are also likely to be the target of T-cell responses. To facilitate the analysis of T-cell responses we have investigated a system that allows expression of these autoantigens as recombinant proteins in autologous cells. Human B-lymphoblastoid cell lines (B-LCL), which are known to present antigen to autologous T cells, were transfected with constructs directing the expression of human TSH-R and TPO. The constructs utilized an expression vector replicating under the control of EBV-derived sequences that is maintained episomally in transfected cells. Both proteins were shown to be expressed by transfected B-LCL and present on the cell surface. Such transfected B-LCL could be used for the derivation, screening and characterization of autologous T-cell clones against thyroid autoantigens.
