Studies On The Quality Control,Safety Evaluation And Stability Of Agents For Gene Therapy

Part Ⅰ Establishment of quality standards and quality control. The "Requirements for Preparation and Control of a Recombinant Adenovirus Ad-HGF" and "Requirements for Preparation and Control of a Recombinant Plasmid pUDKH" were worked out for the sake of control of their quality of both biologies according to the Requirements. 19 items of examinations for Ad-HGF were carried out, including physical properties, sterility test, pyrogen test, examination of bacterial endotoxin content, viral partical count, determination of viral purity, determination of infectivity titer of the virus (pfu/ml), determination of specific titer of the virus, expression of target gene(HGF), determination of replicative adenovirus, residual antibiotics (penicillin, streptomycin) tests, residual cesium test, abnormal toxicity test, residual bovine albumin detection(ng/ml), adenovirus protein identification(cytopathic effect method), determination of molecular weight of adenovirus DNA, enzymatic cleavage profile display,detection of characteristic gene domain E2B. For plasmid pUDKH, 17 items were examined, including physical properties, sterility test, pyrogen test, mycoplasm detection,pH value determination, DNA content determination, DNA purity test, residual RNA detection, residual bacterial genomic DNA detection, residual host bacterial protein detection, content of bacterial endotoxin determination, enzymatic cleavage profile display, analysis of DNA sequence of the target gene, target gene identification test, target gene activity test, abnormal toxicity test, residual Triton X-100 detection, and residual antibiotics (kanamycin) detection. The results showed that the quality of the two biological products were in accord with their Requirement, respectively.Part Ⅱ Evaluation of safty of plasmid pUDKH. (l)The results of acute toxicity test for pUDKH demonstrated that no evident toxicity reactions were found during the observation period for i.m or i.p injection of pUDKH to mice. The MTD of a single i.m. injection in mice was 36.6mg/kg(female mice) or 35.6 mg/kg(male mice); The MTD of i.p injection of pUDKH once in mice was 39.0mg/kg(female mice) or 39 0 mg/kg(male mice).The MTD of injected pUDKH to mice was corresponding to 120-136 times(calculated according to body weight) or 10-11 times(calculatedaccording to body surface area) as of the proposed clinical dosage. (2) Chronic toxicity experiments of plasmid pUDKH were performed in Beagle dogs and rats. The doses of i.m. pUDKH to mice were 0.63 mg/kg and 2.5 mg/kg, corresponding to 22- and 88-fold of the proposed clinical dosage, respectively. For Beagle dogs, the doses of i.m. pUDKH were 0.15 mg/kg,0.375 mg/kg and 0.937 mg/kg, corresponding to 5.3-,13.5- and 31.6-fold of the proposed clinical dosage, respectively. The results demonstrated that no abnormal change was found among various dosage groups during administration and recovery by examining general reaction to the drug, peripheral blood cells, serum biochemical parameters, urine biochemical parameters, and histopathologic examination of main organs and tissues. Antibody against HGF in serum was not detectable at each time point. In addition, distribution of target gene HGF was detected in various tissues, and the results showed that high expression of HGF was only detectable in muscles at the local injection site, but was not in other tissues, demonstrating that HGF could be express locally after injection pUDKH into muscles.(3) General pharmacological test was done in mice and dogs in vivo and ileum isolated from guinea pig ex vivo to observe the general pharmacological effects of plasmid pUDKH. The results demonstrated that there were not evident effects on general behavior, activity, mental state, the central nervous system, autonomic nervous system, digestive system of mice after i.m. pUDKH, and it showed not analgesic and hypnotic actions.There were also no remarkable effect on blood pressure, heart rate, ECG parameters, respiration, and body temperature of dogs.Part Ⅲ Evaluation of safety of recombinant adenovirus Ad-HGF. (l)The results of acute toxicity test for Ad-HGF demonstrated that no evident toxicity reactions were found during the observation period after i.m or i.p. injection of Ad-HGF to mice. The MTD of i.m. Ad-HGF once in mice was 1.46× 109 PFU/kg (female mice) or 1.49× 109 PFU/kg(male mice). The figures for i.p Ad-HGF once in mice were 1.45×109 and 1.50× 109 respectively. The MTD of injection Ad-HGF to mice correspond to 190- (calculated according to body weight) or 17.4-fold (calculated according to body surface area) of the proposed clinical dosage. (2) Chronic toxicity experiments of the recombinant adenovirus were carried out in beagles and rats. The doses of i.m. Ad-HGF to rats were 2×108PFU/kg and 1×109 PFU/kg, corresponding to 26- and 130-fold of the proposed clinical dosage, respectively. For beagles, the doses of i.m. Ad-HGF were 2.4×107 pfu/kg and2.4×108 , corresponding to 3.4- and 34-fold clinical dosage, respectively. Ad-HGF was administered 14 times per week for successive 7 weeks. The results demonstrated that there was no abnormal change in beagles of these two groups during administration and recovery period. Antibody against the adenovirus in serum was detectable after the seventh time of i.m. injection to rats, and the antibody vanished at the 13th or 14 th week after of Ad-HGF withdrawal; For beagles, the antibody was detectable in serum at the fourteenth time of i.m. injection successively, and vanished at the 12th week after withdrawal. Antibody against HGF in serum was not detectable at all time points. In addition, HGF could be expressed locally in muscles after injection of Ad-HGF.Part Ⅳ Evaluation of stability of recombinant adenovirus Ad-HGF. To investigate the effect of changes in temperature on storage time of the recombinant adenovirus carrying hepatocyte growth factor (Ad-HGF), Ad-HGF and Ad-GFP in Hank's solution containing 10% glycerin were stored at different temperatures (-60 ℃,-20℃,4℃,25℃ and 37℃) for different times. Then the viral infectivity, gene expression and transfection efficiency of the samples were detected by cytopathogenic effect (CPE), ELISA and flow cytometry methods. The results showed that there was little reduction of all the three parameters at -60℃ for 44 months and at -20℃ for 12 months. These parameters reduced in unremarkable degrees after stored at 4℃ in refrigerator for 2 months, however, after 7 months, there was a more rapid decline in virus titer, gene expression and transfection efficiency. Storing at 25℃ and 37"C, the level of gene expression and transfection efficiency was decreased day by day except for infection titer, suggesting that the activity of recombinant adenovirus is stable when stored at below -20 ℃.