RESUMO
OBJECTIVE: To establish the quality control methods and reference standards for clinical-grade recombinant adenovirus products for human gene therapy. METHODS: The Infectivity of clinical grade recombinant adenoviral vectors is determined by a TCID(50) assay. The purity is determined by a high-performance liquid chromatography (HPLC) assay. A549 cells were used in replication competent adenovirus (RCA) assay of samples by observation of the cytopathic effect. Other quality control assays were performed in accordance with the SFDA Regulations for Biological Products. RESULTS: The recombinant adenovirus encoding human p53 gene produced at SiBiono (Lot 20010701) has the following quality attributes: viral particle concentration: 1.03 (10(12) VP/ml, infectivity titer: 5.01 (10(10) IU/ml, specific infectivity (IU/VP): 4.86%, higher than the 3.3% required by the Food and Drug Administration (FDA), USA; Purity by HPLC analysis: 98.62%, higher than the 95% purity specified by SFDA; and level of RCA: less than 1 RCA/3 (10(10) VP, meeting the standards established by SFDA. CONCLUSIONS: A whole set of quality standards of clinical-grade recombinant adenovirus vectors has been established so as to ensure the clinical safety and efficacy of recombinant adenoviral vectors for human gene therapy.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Proteínas Recombinantes de Fusão/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Terapia Genética/normas , Vetores Genéticos/genética , Vetores Genéticos/normas , Humanos , Controle de Qualidade , Proteínas Recombinantes de Fusão/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Receptor protein tyrosine kinases (RPTKs) play important roles in the regulation of a variety of cellular processes including cell migration, proliferation, and protection from apoptosis. Here, we report the identification and characterization of a novel RPTK-like molecule that has a critical role in induction of tumorigenesis and metastasis and is termed Novel Oncogene with Kinase-domain (NOK). NOK contains a putative single transmembrane domain and a conserved intracellular tyrosine kinase domain that shares homology with members of the platelet-derived growth factor/fibroblast growth factor receptor superfamily. NOK was exclusively located in the cytoplasm. NOK mRNAs were detected in limited human organs and expressed with the highest abundance in the prostate. A variety of tumor cells also expressed the NOK mRNAs. We demonstrated that NIH3T3 and BaF3 cells could be strongly transformed by the expression of the NOK gene as examined by colony formation experiment. In addition, BaF3 cells with the stable expression of NOK induced rapid tumorigenesis in nude mice. Interestingly, these NOK-expressing tumor cells could promptly invade and spread into various distinct organs and form metastatic foci, eventually leading to the rapid death of these animals. Moreover, molecular mechanism studies indicated that NOK could concomitantly activate both MAP kinase and phosphatidylinositol 3'-kinases (PI3K) pathways in stable BaF3 cells. Thus, our results both in vitro and in vivo suggest that NOK is a novel oncogene with the capacity of promoting cell transformation, tumorigenesis, and metastasis.