Objective: To establish and validate a biological activity detection method for detecting the transcription level of rAAV2-ND4 injection. Methods: rAAV2-ND4 sample (3.0×1010 vg·mL-1 at 360 μL per well) was infected with HEK293T cells (3.0×105 cells·mL-1, 0.8 mL per well, pre-cultured at 37 ℃ with 5% carbon dioxide for 24 h), followed by 2 h of culture. An appropriate amount of culture medium was added and continued to culture for 24 h, then the cells were collected. Cell total RNA was extracted according to the operating instructions of the cell total RNA extraction kit (cell lysis, adsorption, deproteinization, impurity removal, and elution). Using the extracted total RNA of cells as a template, the Ct values of mRNA and β-actin internal reference gene of ND4 were detected using qPCR method. By simultaneously setting up rAAV2-ND4 reference samples for parallel testing,the relative biological activity of rAAV2-ND4 was detected using double calibration detection with β-actin internal reference genes and the rAAV2-ND4 reference substance. The methodological validation included accuracy, precision, linearity, and specificity. The inter batch consistency of the three products was also investigated. Results: The results of qPCR detection of ND4 mRNA transcription and reference correction method indicated a dose-response relationship between ND4 mRNA and rAAV2 ND4 infect titers. According to the verification of methodology, the deviation rate between the detection value and the expected value of the method was between 100% and 130%. The RSD of 9 results for different personnel at different times was 13.75%. The method was linearity from 1.0×1010to 5.0×1010 vg·mL-1 of rAAV2-ND4 titer, r>0.97. The method had good specificity. The RSD of the three batches of rAAV-ND4 detection values was 9.3%. Conclusion: A preliminary biological activity detection method for rAAV2 ND4 transcription level has been established.
QIN Xi, CHEN Long, SHI Xin-chang, YANG Jing-qing, PAN Yan-qun, YU Lei, BI Hua, PEI De-ning, AN Yi-fang, PAN Yue, LI Xiang, ZHOU Yong
. Biological activity detection method for gene transcription level of rAAV2-ND4 injection*[J]. Chinese Journal of Pharmaceutical Analysis, 2023
, 43(11)
: 1820
-1825
.
DOI: 10.16155/j.0254-1793.2023.11.02
[1] FARRAR GJ, CHADDERTON N, KENNA PF, et al. Mitochondrial disorders: aetiologies, models systems, and candidate therapies[J]. Trends Genet, 2013, 29(8): 488
[2] JANKAUSKAITÉ E, BARTNIK E, KODRO A. Investigating Leber’s hereditary optic neuropathy: cell models and future perspectives[J]. Mitochondrion, 2017, 32(1): 19
[3] YU H, KOILKONDA RD, CHOU TH, et al. Gene delivery to mitochondria by targeting modified adenoassociated virus suppresses Leber’s hereditary optic neuropathy in a mouse model[J]. Proc Natl Acad Sci USA, 2012, 109(20): 1238
[4] HüSER D, GOGOL-DÖRING A, LUTTER T, et al. Intergration preferences of wildtype AAV-2 for consensus rep-binding sites at numerous loci in the human genome[J]. PLoS Pathog, 2010, 6(7):e1000985
[5] SCHULTZ BR,CHAMBERLAIN JS. Recombinant adeno-associated virus transduction and integration[J]. Mol Ther, 2008, 16(7):1189
[6] CARTER BJ. Adeno-associated virus and the development of adeno-associated virus vectors: a historical perspective[J]. Mol Ther, 2004, 10(6):981
[7] PENAUD-BUDLOO M, LE GUINER C, NOWROUZI A, et al. Adeno-associated virus vector genomes persist as episomal chromatin in primate muscle[J]. J Virol, 2008, 82(16):7875
[8] VAN VLIET KM, BLOUIN V, BRUMENT N, et al. The role of the adeno-associated virus capsid in gene transfer[J]. Methods Mol Biol, 2008, 437: 51
[9] MUELLER C, GERNOUX G, GRUNTMAN AM, et al. 5 year expression and neutrophil defect repair after gene therapy in alpha-1 antitrypsin deficiency[J]. Mol Ther, 2017, 25(6):1387
[10] RANGARAJAN S, WALSH L, LESTER W, et al. AAV5-factor VIII gene transfer in severe hemophilia A[J]. N Engl J Med, 2017, 377(23): 2519
[11] GEORGE LA, SULLIVAN SK, GIERMASZ A, et al. Hemophilia B gene therapy with a high-specific-activity factor IX variant[J]. N Engl J Med, 2017, 377(23):2215
[12] BENNETT J, WELLMAN J, MARSHALL KA, et al. Safety and durability of effect of contralateral-eye administration of AAV2 gene therapy in patients with childhood-onset blindness caused by RPE65 mutations: a follow-on phase 1 trial[J]. Lancet, 2016, 388(10045):661
[13] RAKOCZY EP, LAI CM, MAGNO AL, et al. Gene therapy with recombinant adeno-associated vectors for neovascular age-related macular degeneration: 1 year follow-up of a phase 1 randomised clinical trial[J]. Lancet, 2015, 386(9990):2395
[14] YANG S, MA SQ, WAN X, et al. Long-term outcomes of gene therapy for the treatment of Leber’s hereditary optic neuropathy[J]. EBio Med, 2016, 10(8):258
[15] WAN X,PEI H,ZHAO M, et al. Efficacy and safety of rAAV2-ND4 treatment for Leber’s hereditary optic neuropathy[J]. Sci Rep, 2016, 6(2):21587
[16] WRIGHT JF. Manufacturing and characterizing AAV-based vectors for use in clinical studies[J]. Gene Ther, 2008, 15(11):840
