RESUMO
PURPOSE: To construct an EGF receptor (EGF-R)-mediated histone H1(0)-based gene delivery system for gene therapy. METHODS: A recombinant DNA containing histone H1(0), EGF-R ligand, and endosomalytic domains was constructed in a prokaryotic vector and expressed in E. coli. Expression of the beta-galactosidase (beta-gal) gene in the tumor cells and tissues was observed after transduction of the beta-gal gene packaged by purified fusion proteins in vitro and in vivo. RESULTS: As an extension of the research on previously reported chemically synthetic composite polypeptide gene delivery systems, this genetically engineered polypeptide has proved to be capable of targeting the beta-galactosidase (beta-gal) gene into EGF-R-positive cancer cells both in vitro and in vivo. We also studied the time course of beta-gal gene expression in tumor tissues delivered in vivo by this polypeptide vector. At 24 h after administration, expression of the beta-galactosidase gene in tumor reached peak levels. The dosage optimization of administered polyplex was also investigated. The optimal dose of polyplex per mouse was 1 microg DNA packaged by 3 microg of composite polypeptide. CONCLUSIONS: The genetically engineered polypeptide based on histone H1(0) is a promising gene delivery system targeting EGF-R.
Assuntos
Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/terapia , Receptores ErbB/metabolismo , Terapia Genética/métodos , Histonas/genética , Osteossarcoma/metabolismo , Osteossarcoma/terapia , Animais , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/genética , Escherichia coli , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Proteínas de Fusão Oncogênica/análise , Proteínas de Fusão Oncogênica/isolamento & purificação , Osteossarcoma/enzimologia , Osteossarcoma/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase , Células Tumorais Cultivadas , beta-Galactosidase/metabolismoRESUMO
OBJECTIVE: To investigate gene transfer efficiency of a novel target non-viral vector GE7 and effects of herpes simplex virus thymidine kinase (HSV(1)-tk)/ganciclovir (GCV) mediated by it in vitro. METHODS: The epidermal growth factor receptor (EGF-R) target gene delivery system GE7 was constructed. Human ovarian cancer cell line CAOV3 was transfected in vitro with beta-galactosidase (beta-gal) as reporter gene and HSV(1)-tk gene as therapeutic gene using this gene delivery system. By means of the assay of X-gal staining, Northern blotting, cell growth-inhibiting curve and so on, the transferring efficiency of exogenous genes and killing effects are observed. RESULTS: It showed that gene transfer efficiency is over 80%. When 10 mg/L GCV was put into ovarian cells transfected with HSV(1)-tk gene, 95% of cells were killed, and the apoptosis ratio reached up to 30. CONCLUSIONS: The GE7 gene delivery system is an effective and safe delivery system. GE7/HSV(1)-tk/GCV therapeutic gene system is appraising for ovarian cancer.