Pharmaceutical Study And Genetherapy Investigation On PUDK-HGF

The recombinant plasmid pUDK-HGF, encoding human hepatocyte growth factor (rHGF), is potential for gene therapy of peripheral arterial occlusion disease. Our preclinical studies showed that pUDK-HGF induced angiogenic effects on the chicken choriollantoic membrane; intramuscular injection of pUDK-HGF into rat or dog hindlimb ischemia models, strong HGF expression was observed in local skeletal muscle tissue , the collateral vessel growth induced by pUDK-HGF were significantly increased in a dose-dependent manner. pUDK-HGF has been submitted to Chinese SFDA for clinic trails.Large scale production of pUDK-HGF is a key step for its application. The process was developed and included fermentation, cell harvesting , alkaline lysis , capturing plasmid DNA with Q-Sepharose XL chromatography, size-exclusion with Sephacry S1000 column and polishing with Source 15Q anion-exchange chromatography. The process was reproducible without using any animal derived enzymes or toxic solvents and suitable for transfering to industrial manufacture of pUDK-HGF.The quality of a pure bulk product was analyzed. The plasmid pUDK-HGF concentration and purity (A260/A280) at 2.01mg/ml and 1. 86 determined by spectrophotometric absorbance at 260nm and A260/A280. The supercoiled DNA homogeneity at 95. 09% and non-visualized RNA on 0. 8% agarose gel analysis; The contents of chromosome DNA < 0.2%, contaminant protein < 1μg/mg plasmid DNA and endotoxin level of < 10EU/mg plasmid DNA were determined by blotting, ELISA and TAL test respectively. The HGF expression of transfected CHO cells at 39.15ng/ml level was assayed using ELISA, expressed HGF significantly induced the migration of endothelial ECV304...