RESUMEN
Renal anemia is predominantly caused by a relative deficiency in erythropoietin (EPO). Conventional treatment for renal anemia includes the use of recombinant human EPO (rhEPO) or a long-acting erythropoiesis-activating agent named darbepoetin alfa, which is a modified rhEPO with a carbohydrate chain structure that differs from native hEPO. We have developed a biosimilar to darbepoetin alfa designated JR-131. Here, we comprehensively compare the physicochemical and biological characteristics of JR-131 to darbepoetin alfa. JR-131 demonstrated similar protein structure to the originator, darbepoetin alfa, by peptide mapping and circular dichroism spectroscopy. Additionally, mass spectroscopic analyses and capillary zone electrophoresis revealed similar glycosylation patterns between the two products. Human bone marrow-derived erythroblasts differentiated and proliferated to form colonies with JR-131 to a similar degree as darbepoetin alfa. Finally, JR-131 stimulated erythropoiesis and improved anemia in rats similarly to darbepoetin alfa. Our data show the similarity in physicochemical and biological properties of JR-131 to those of darbepoetin alfa, and JR-131 therefore represents a biosimilar for use in the treatment of renal anemia.
Asunto(s)
Biosimilares Farmacéuticos/farmacología , Darbepoetina alfa/farmacología , Eritropoyesis/efectos de los fármacos , Anemia/tratamiento farmacológico , Animales , Células CHO , Cricetinae , Cricetulus , Darbepoetina alfa/química , Modelos Animales de Enfermedad , Electroforesis Capilar , Glicosilación/efectos de los fármacos , Riñón/patología , Masculino , Peso Molecular , Nefrectomía , Mapeo Peptídico , Estructura Secundaria de Proteína , Ratas Sprague-Dawley , Azúcares/análisis , Resultado del TratamientoRESUMEN
A new multifunctional gene delivery system was constructed with diphtheria toxin's functional domains. Used functional domains are T domain for endosomal escape and R domain for efficient internalization into cell. In order to conjugate these domains into PEI polyplex, diphtheria toxin T and R domains-streptavidin fusion protein (DTRS) was prepared. The conjugation of the DTRS with biotinylated PEI polyplex (DTRS-polyplex) lead to the significant enhancement of transfection efficiency when compared with plain PEI/pDNA polyplex in CHO-K1 cell. It was demonstrated that DTRS-polyplex had high endosomal escape efficiency and internalization efficiency by several measurements, such as in vitro intracellular trafficking observation and the internalization inhibition with several inhibitors. These results suggest that this multifunctional non-viral vector may contribute to the future cancer gene therapy.
Asunto(s)
Toxina Diftérica/química , Técnicas de Transferencia de Gen , Polietileneimina/química , Transfección/métodos , Animales , Transporte Biológico , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , ADN/administración & dosificación , Toxina Diftérica/metabolismo , Endosomas/metabolismo , Terapia Genética/métodos , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Espacio Intracelular/metabolismo , Plásmidos , Polietileneimina/metabolismoRESUMEN
The endosomal escape is a well-known serious obstacle for non-viral gene delivery. This is because of an acidic and enzymatic degradation of the contents of the endosome/lysosome. Therefore, the internalized gene needs to be efficient released into the cytosol to obtain the efficiently transfection efficiency. On the other hand, the diphtheria toxin T domain fuses with endosome membrane by pH decrease, then enhances the endosomal escape of the diphtheria toxin C fragment. In this study, we constructed diphtheria toxin T domain-conjugated poly(ethylenimine)s (PEI) polyplex for enhancing the endosomal escape of exogenous gene. The conjugation of diphtheria toxin T domain with PEI/pDNA polyplex leads to the significant enhancement of transfection efficiency when compared with plain PEI/pDNA polyplex. The pH-responsive increase in hydrophobicity of the diphtheria toxin T domain might not only trigger the perturbation of the endocytic vesicle membrane but might also increase the membrane permeability.
Asunto(s)
Toxina Diftérica/química , Toxina Diftérica/metabolismo , Endosomas/metabolismo , Vectores Genéticos/genética , Polietileneimina/metabolismo , Transfección/métodos , Animales , Transporte Biológico , Biotinilación , Células COS , Permeabilidad de la Membrana Celular , Chlorocebus aethiops , Endocitosis , Humanos , Concentración de Iones de Hidrógeno , Espacio Intracelular/metabolismo , Liposomas/metabolismo , Estructura Terciaria de Proteína , Estreptavidina/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
In order to enhance the internalization of exogenous gene and add cell specificity to non-viral vectors, receptor-binding elements have been widely utilized to mimic the virus infection. Herein, for the purpose of intensifying the effects of the ligand on gene delivery, dual receptor-binding elements, transferrin (Tf) and transforming growth factor alpha (TGFalpha), were introduced into the polyethyleneimine polyplex. The transfection and internalization efficiency by dual Tf- and TGFalpha-introduced polyplex (Tf&TGFalpha-polyplex) was examined in A549 and CHO-K1 cells, respectively. In A549, Tf&TGFalpha-polyplex had higher transfection efficiency when compared to that by single Tf- or TGFalpha-introduced polyplex (Tf-polyplex and TGFalpha-polyplex), respectively, while no enhancement was observed in CHO-K1. Moreover, in A549, the internalization efficiency of dual Tf&TGFalpha-polyplex was higher than that of single Tf- and TGFalpha-polyplex. In contrast, in CHO-K1, no difference in internalization efficiency was observed. In the presence of excess free transferrin or TGFalpha, the internalization efficiency of Tf&TGFalpha-polyplex was strongly inhibited only in A549, not in CHO-K1. In summary, the enhancement of internalization efficiency by dual ligands is an important factor for improving transfection efficiency. In addition, the effect of dual ligands depends on cell species; receptor-mediated and efficient internalization may be related to this enhanced transfection efficiency.
Asunto(s)
Técnicas de Transferencia de Gen , Iminas/química , Polietilenos/química , Transferrina/farmacología , Factor de Crecimiento Transformador alfa/farmacología , Animales , Biotinilación , Células CHO , Bovinos , Línea Celular Tumoral , Cricetinae , Cricetulus , ADN/química , ADN/metabolismo , Receptores ErbB/metabolismo , Genes Reporteros , Vectores Genéticos , Humanos , Iminas/metabolismo , Ligandos , Luciferasas/metabolismo , Peso Molecular , Polietilenos/metabolismo , Receptores de Transferrina/metabolismo , Albúmina Sérica Bovina/metabolismo , TransfecciónRESUMEN
A chitosan derivative, 6-amino-6-deoxy chitosan (6ACT), was galactosylated and was investigated as a gene carrier. A series of galactose-modified 6ACT (Gal-6ACT) with degrees of substitution (d.s.) ranging from 3% to 50% per pyranose were prepared by reductive alkylation with lactose. DNA retardation assays showed that the electrostatic interaction between Gal-6ACT and plasmid DNA was not changed by galactose modification up to 50% per pyranose of 6ACT. Gal-6ACT with a d.s. of 38% was bound to galactose-recognizing lectin, RCA120. A significant increase in transfection efficiency for HepG2 cells was observed at degree of substitutions ranging from 18% to 50% and at N/P values ranging from 1.5 to 2.5. Under optimum conditions, Gal-6ACT showed about 10 times higher efficiency than 6ACT. However, a slight uptake by the galactose receptors on hepatocytes was observed by flow cytometric analysis. Moreover, Gal-6ACT with a d.s. of 38% mediated efficient gene transfer into both A549 and HeLa cells lacking the galactose receptor. These results suggest that the enhancement of transfection efficiency of Gal-6ACT was not due to the increase of receptor-mediated cellular uptake. In addition, the enhanced gene transfer efficiency was not specific to the galactose modification because the efficiency of glucose-modified 6ACT for HepG2 cells was similar as that of Gal-6ACT.