Valuation of the safety of recombinant adenovirus coding human IL-12 protein on mice

To evaluate the safety of recombinant adenovirus coding the human IL-12 protein in mice. Subjects and methods: BALB/c mice were divided into 3 groups, each group of 9 mice, including control group, recombinant adenovirus coding the human IL-12 protein injection group at 106 vp/head, recombinant adenovirus coding the human IL-12 protein injection group at 107 vp/head.

Journal of military pharmaco-medicine no8-2019 EVALUATION OF THE SAFETY OF RECOMBINANT ADENOVIRUS CODING HUMAN IL-12 PROTEIN ON MICE Nguyen Thai Bieng1; Hoang Quoc Truong2; Dang Quang Chung3; Ho Anh Son SUMMARY Objectives: To evaluate the safety of recombinant adenovirus coding the human IL-12 protein in mice Subjects and methods: BALB/c mice were divided into groups, each group of mice, including control group, recombinant adenovirus coding the human IL-12 protein injection group at 10 vp/head, recombinant adenovirus coding the human IL-12 protein injection group at 10 vp/head Mice were monitored for general health, liver and kidney function, and tested for the presence of recombinant adenovirus coding the human IL-12 protein in mouse organs Results: Recombinant adenovirus coding the human IL-12 protein injected mice were healthy, no dead mice Hepatic and renal function showed no significant difference through ALT, AST, bilirubine, urea and creatinine Recombinant adenovirus coding the human IL-12 protein was not found in rat organs by PCR technique Conclusion: Recombinant adenovirus coding the human IL-12 protein is safe for mice at dose of 10 vp/head * Keywords: Adenovirus; IL-12; Recombinant adenovirus coding the human IL-12 protein; Mice INTRODUCTION Interleukine-12 (IL-12) is one of the promising cytokines in anti-cancer immunotherapy IL-12 activates and triggers type support T cells (Th1), stimulates differentiation of toxic T cells (TCD8+); promotes T-cells to maintain the production of interferon-gamma (IFN-γ) which is necessary for tumor suppression; directly affecting natural killer cells to kill cancer In addition, IL-12 also has an indirect anti-vascular genesis through intermediate proteins such as IP-10 and Mig induced by IFN-γ The recombinant adenovirus vector coding IL-12 protein is a product of the national project "Researching on the application of the interleukine-12 protein for the treatment of hepatocellular carcinoma", integrating the 3rd generation Ad vector and IL-12-encoded gene has been shown to transfer into liver cancer cells, as well as produce IL-12 in vitro To be able to use in the next study, we conducted an assessment of safety of this preparation on experimental animals SUBJECTS AND METHODS Subjects - BALB/c mice were kept in a laboratory, cared as guidance at the Institute of Biomedicine and Pharmacy, Vietnam Military Medical University Vietnam Military Medical University 108 Military Central Hospital Viet Tiep Hospital Corresponding author: Ho Anh Son (hoanhsonhp@gmail.com) Date received: 19/09/2019 Date accepted: 18/10/2019 120 Journal of military pharmaco-medicine no8-2019 - Recombinant adenovirus coding IL12 protein (Ad-IL12) and recombinant adenovirus (Ad), provided by Department of Molecular Biology, 108 Military Central Hospital Material and equipment - Group (control): sodium chloride 0.9% x 0.20 mL/mouse, tail vein injection, single dose * Assessment of safety of Ad-IL12: - Body condition, body weight, death, stool and other abnormalities Primer pORF-IL12 forward, 5’-TGGGAG TACCCTGACACCT-3’ and pORF-IL12 reverse, 5’-GTACCCCTACTCCAGGAAC3’ for IL-12 detection - Assess liver and kidney function: Determine the activity/concentration of AST, ALT, total bilirubine, urea, creatinine in serum (1 week after injection) Gel-Doc & Dolphin (gel) scanner, electrophoresis kit (Bio-RAD), PCR 9700 (Applied BioSystems), centrifuge (Beckman), spectrophotometer (Beckman), thermal block machine (Eppendorf) - Discover genetic material of Ad-IL12 in mouse organs by PCR (after weeks of injection) PCR reaction was optimized as follow: 12.5 mcL 2X PCR Master mix; 10 pM forward and reverse bait; mcL total DNA; add sterile water to 25 mcL The thermal cycle for PCR: 95oC, minutes; 35 cycles [95oC, 20 seconds; 58oC, 30 seconds; 72oC, minute]; 72oC, minutes; keep heat at 4oC The product of PCR reaction was captured on 1% agarose gel Research methods The mice were randomly divided into groups, mice per group: -.Group 1: Ad-IL12 at dose of 106 vp/ mouse, tail vein injection, single dose - Group 2: Ad-IL12 at dose of 107 vp/ mouse, tail vein injection, single dose RESULTS Body condition of mice during the experiment After Ad-IL12 administration, the mice were still eating and operating normally, agile, smooth hair, clear eyes, and dry anus There were no signs of abnormalities such as ruffled feathers, bleeding, loose movement, seizures, etc No mice die was observed Table 1: Mice body weight Group Body weight (g) n Day Day 22.4 ± 1.12 23.2 ± 0.88 Group (10 vp/head) 22.1 ± 1.15 22.8 ± 0.96 Group (control) 22.3 ± 1.28 23.2 ± 1.0 Group (10 vp/head) The mice still gained weight during the experiment Thus, Ad-IL12 at the two studied dose levels did not affect the systemic status of the mice 121 Journal of military pharmaco-medicine no8-2019 Results of biochemical tests of liver and kidney function Table 2: Results of AST activity in mice Group n AST activity (U/L) Group (10 vp/head) 195.5 ± 26.87 Group (10 vp/head) 176.9 ± 33.23 Group (control) 187.1 ± 14.48 p > 0.05 Ad-IL12 at both dose levels (106 and 107 vp/mouse) slightly increased blood AST enzyme activity in mice However, there was no statistically significant difference in blood activity of AST enzyme (U/L) among study mice groups Table 3: Results of ALT activity in study mice Group n ALT activity (U/L) Group (10 vp/head) 33.0 ± 5.57 Group (10 vp/head) 43.3 ± 27.5 Group (control) 27.7 ± 8.21 p > 0.05 The serum ALT (U/L) activity was kept in normal limits in three groups Thus, Ad-IL12 at the two studied dose levels (106 and 107 vp/mouse) did not affect the mice serum ALT activity Table 4: Results of total bilirubine in study mice Group n Total bilirubine (mcmol/L) Group (10 vp/head) 33.7 ± 14.75 Group (10 vp/head) 51.0 ± 19.87 Group (control) 41.9 ± 16.67 p > 0.05 The difference in total serum bilirubine in the experimental mice was not statistically significant compared to the control group Table 5: Results of urea concentration in study mice serum n Urea (mmol/L) Group 6.7 ± 1.67 Group (10 vp/ head) 7.2 ± 1.85 Group (control) 6.3 ± 1.06 Group (10 vp/head) p > 0.05 The urea concentration in experimental groups was within normal limits Thus, Ad-IL12 injection did not have significant effect on urea concentration in experimental mice 122 Journal of military pharmaco-medicine no8-2019 Table 6: Results of creatinine concentration in study mice serum Group n Creatinine (mcmol/L) 6 45.1 ± 8.72 Group (10 vp/head) 36.9 ± 12.8 Group (control) 48.7 ± 11.7 Group (10 vp/head) p > 0.05 The creatinine concentration in experimental group was within normal limits Thus, Ad-IL12 injection did not have significant effect on creatinine concentration in experimental mice Results of PCR product amplifying IL-12 specific gene in mouse organs after intravenous Ad-IL12 injection Figure 1: The results amplify IL-12 gene segments from total DNA extracted from organ samples of mice (Group: (A), (B) and control group; liver (G), heart (Ti), striated muscle (Muscle), lung (P), brain (N), kidney (Th) and serum (M); samples (-), negative PCR; sample (+), positive plasmid) 123 Journal of military pharmaco-medicine no8-2019 No human gene segment encoding IL-12 was detected in organs: heart, liver, lung, kidney, brain, skeletal muscle, serum in mouse groups injected with Ad-IL12, compared with non-injected control group Thus, Ad-IL12 with injection doses did not develop, replicated in mouse tissues and organs DISCUSSION Ad-IL12 does not alter biochemical indices that reflect renal and hepatic function The mice that were injected with Ad-IL12 were still functioning normally, not dead and still growing steadily Amplified reactions for the gene encoding IL-12 was not detected in the heart, liver, lungs, kidneys, brain, striated muscle, and serum in Ad-IL12 injected mice When injecting animals with early generation Ad vector (1, 2) results in late toxicity, occurring day to several weeks due to the low level expression of viral proteins from the vector backbone With 3rd generation Ad (Helper-dependent Adenovirus, HDAd), toxicity appeared only 24 - 48 hours after injection due to absence of virus expression genes The use of HDAd has brought advantages in therapy, while limiting the toxicity of many toxic gene segments on adenovirrus With the purpose of designing to optimize the immune response and further improve the safety of the Adenovirus vector, the HDAd vector system was generated The designed vector was removed all gene sequences carrying the virus code (ie, eliminate all virulent genes 124 of the virus) The entire deleted gene sequence of the virus will be replaced by a gene carrier design or a non-coding DNA sequence called "stuffing", to keep the vector of the appropriate size for packaging The elimination of viral genes strongly reduced the host's cytotoxic response [1, 2, 3] A study by Ng et al showed that this third-generation vector was safer and the toxicity significantly decreased when high doses were used in mice [4] In addition, low-level lipoprotein receptors using HDAd vectors have been produced for long periods to combat atherosclerosis in rat models with a history of hypercholesterolemia that have been mentioned in Nomura et al’s study [5] In addition, this vector is also used in ex vivo technology by introducing the cystic fibrosis gene through the sweat glands into the human body for high gene transfer efficiency [6] The production of recombinant Helperdependent Adenovirus virus carrying and transmitting therapeutic genes that can infect target cells with high efficiency, good quality eliminates the contamination of the helper virus being further optimized to have get recombinant virus products that carry safe therapeutic genes CONCLUSSION Administration of Ad-IL12 at doses of 10 and 107 vp/mouse did not cause changes in kidney and liver function AdIL12 was undetected in mice organs by PCR technique Journal of military pharmaco-medicine no8-2019 REFERENCES He X.X, Chang Y, Meng F.Y, Wang M.Y, Xie Q.H, Tang F, Li P.Y, Song Y.H, Lin J.S MicroRNA-375 targets AEG-1 in hepatocellular carcinoma and suppresses liver cancer cell growth in vitro and in vivo Oncogene 2012, 31, pp.3357-3369 malignancies Clin Cancer Res 2002, (11), pp.3383-3393 P.P Cre levels limit packaging signal excision efficiency in the Cre/loxP Helperdependent Adenoviral vector system Journal of Virology 2002, 76, pp.4181-4189 Nicola Brunetti-Pierri et al Helperdependent Adenoviral vectors for liver-directed gene therapy Hum Mol Genet 2011, 20 (1), pp.1-28 Nomura S Low-density lipoprotein receptor gene therapy using helper-dependent Adenovirus produces long-term protection against atherosclerosis in a mouse model of familial hypercholesterolemia Gene Therapy 2004, 11, pp.1540-1548 Robertson M.J et al Interleukine 12 immunotherapy after autologous stem cell transplantation for hematological Palmer D et al Improved system for helper-dependent adenoviral vector production Molecular Therapy 2003, 8, pp.846-852 125 ... advantages in therapy, while limiting the toxicity of many toxic gene segments on adenovirrus With the purpose of designing to optimize the immune response and further improve the safety of the Adenovirus. .. the contamination of the helper virus being further optimized to have get recombinant virus products that carry safe therapeutic genes CONCLUSSION Administration of Ad-IL12 at doses of 10 and 107... sequence of the virus will be replaced by a gene carrier design or a non -coding DNA sequence called "stuffing", to keep the vector of the appropriate size for packaging The elimination of viral