目的:建立并验证重组5型腺相关病毒(recombinant adeno-associated virus type 5,rAAV5)基因组滴度测定的数字PCR方法,并对微滴数字PCR方法和芯片数字PCR方法进行比较。方法:分别用不同浓度的SDS和EDTA前处理 r AAV5样品,用微滴数字PCR测其基因组滴度,对病毒样品前处理试剂进行优化;在其他试验条件不变的情况下,分别设置微滴数字PCR的退火温度为60、58、54、52 ℃,以WPRE引物扩增,比较扩增效果,对数字PCR的退火温度进行优化;分别采用微滴数字PCR和芯片数字PCR测定rAAV5的基因组滴度并比较。结果:终浓度为1% SDS和62.5 mmol·L-1 EDTA按1∶1(v/v)的比例混合后前处理rAAV5,测定的基因组拷贝数较高,前处理液裂解效果较好;当退火温度为54 ℃时,微滴数字PCR中阳性微滴和阴性微滴分离效果最好,扩增特异性最强;2种测定结果的试验室内精密度和回收率接近,测定同一裂解样本的结果较为接近(在1个数量级)。结论:初步建立了数字PCR方法并验证,为后续的进一步验证奠定基础。
郑红梅, 秦玺, 李永红, 于雷, 毕华, 饶春明, 朱留强, 杨靖清, 史新昌, 周勇
. rAAV5基因组滴度测定的数字PCR方法研究*[J]. 药物分析杂志, 2023
, 43(11)
: 1826
-1832
.
DOI: 10.16155/j.0254-1793.2023.11.03
Objective: To establish and verify a digital PCR method for the determination of recombinant adeno-associated virus type 5(rAAV5) genome titer, and to compare the performance of chip and droplet digital PCR methods. Methods: By examine the different lysing outcome from various concentrations of SDS or EDTA, the adeno-associated virus lysis method was optimized. To ensure other experimental conditions being consistent, the rAAV5 sample was amplified with WPRE primers in the droplet digital PCR method, and the annealing temperature was set to 60, 58, 54, 52 ℃. With amplification effect compared, the annealing temperature was optimized. Both chip and droplet digital PCR were tested to determine the genomic titer of rAAV5, and the consistency of the results of the two methods was compared. Results: The final concentration of 1% SDS and 62.5 mmol·L-1 EDTA was mixed 1∶1(v/v) and then rAAV5 was lysed. The measured genome copy number was high, the lysing outcome was good. At annealing temperature 54 ℃, the positive droplets in the droplet digital PCR and the separation effect of negative droplets was ideal, and the amplification specificity is the strongest. Comparing the two methods, it was found that the laboratory precision and recovery rates of the two determination results are similar, and the results of determining the same pyrolysis sample were relatively close (on the same order of magnitude). Conclusion: A preliminary digital PCR method has been established and validated, laying the foundation for further validation in the future.
[1] 吴剑卿,许伟,李燕.腺相关病毒载体在肺疾病基因治疗中的应用[J]. 国际呼吸杂志,2007,7(10):777
WU JQ, XU W, LI Y. Application of adeno-associated virus vector in gene therapy of lung diseases [J]. Int J Respir, 2007, 7 (10): 777
[2] GOROVITS B, AZADEH M, BUCHLIS G, et al. Evaluation of the humoral response to adeno-associated virus-based gene therapy modalities using total antibody assays[J]. AAPS J, 2021, 23(6):108
[3] XIE Q, BU W, BHATIA S, et al. The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy[J]. Proc Nati Acad Sci USA, 2002, 99(16): 10405
[4] OGDEN PJ, KELSIC ED, SINAI S, et al. Comprehensive AAV capsid fitness landscape reveals a viral gene and enables machine-guided design.[J]. Science, 2019, 366(6469): 1139
[5] KEELER AM, FLOTTE TR. Recombinant adeno-associated virus gene therapy in light of luxturna (and Zolgensma and Glybera): where are we, and how did we get here[J]. Annu Rev Virol, 2019, 6(1):601
[6] DARROW JJ. Luxturna: FDA documents reveal the value of a costly gene therapy[J]. Drug Discov Today, 2019, 24(4):949
[7] 夏训明.美国FDA批准一种基因疗法药物治疗RPE65双等位基因突变型遗传性视网膜变性病[J]. 广东药科大学学报,2017,33(6):752
XIA XM. A gene therapy drug approved by the US FDA for the treatment of RPE65 double allele mutant hereditary retinal degeneration [J]. J Guangdong Pharm Univ, 2017, 33(6): 752
[8] 夏训明.美国FDA批准Zolgensma(onasemnogene abeparvovec-xioi)治疗儿童脊髓性肌萎缩症[J]. 广东药科大学学报,2019,35(3):332
XIA XM. Zolgensma (onasemnogene abeparvovec-xioi) approved by FDA in the treatment of spinal muscular atrophy in children [J]. J Guangdong Pharm Univ, 2019, 35 (3): 332
[9] MILLER NE. Glybera and the future of gene therapy in the European Union[J]. Nat Rev Drug Discov, 2012, 11(5): 419
[10] 徐洋,闫荟羽,陶娌娜,等.基因治疗药物Alipogene tiparvovec的研究进展[J]. 中国药房,2015,26(11):1579
XU Y, YAN HY, TAO LN, et al. Progress in the research of gene therapy drug Alipogene tiparvovec [J]. J Chin Pharm, 2015, 26 (11): 1579
[11] 李永红,毕华,秦玺,等.基因治疗产品的质量控制分析方法及研究进展[J]. 药物分析杂志,2020,40(1):4
LI YH, BI H, QIN X, et al. Quality control analysis methods and research progress of gene therapy products [J]. Chin J Pharm Anal, 2020, 40 (1): 4
[12] 王晓,黄晓平,黎玲,等.重组腺相关病毒基因药物三种滴度的比较与分析[J]. 华侨大学学报(自然科学版),2021,42(4):507
WANG X, HUANG XP, LI L, et al. Comparison and analysis of three titers of recombinant adeno-associated virus gene drugs [J]. J Huaqiao Univ (Nat Sci Ed), 2021, 42 (4): 507
[13] VELDWIJK, M. Development and optimization of a real-time quantitative PCR-based method for the titration of AAV-2 vector stocks[J]. Mol Ther, 2002, 6(2):272
[14] MAYGINNES JP, REED SE, BERG HG, et al. Quantitation of encapsidated recombinant adeno-associated virus DNA in crude cell lysates and tissue culture medium by quantitative,real-time PCR[J]. J Virol Methods, 2006, 137(2):193
[15] SANMIGUELV J, GAO G, VANDENBERGHE LH. Quantitative and digital droplet-based AAV genome titration: design and delivery[J]. Methods Mol Biol, 2019, 1950:51
[16] 姜志军,江颖,徐摇光,等.利用微滴数字PCR方法快速分析转基因玉米中外源基因的拷贝数[J]. 生物技术进展,2016,6(4):288
JIANG ZJ, JIANG Y, XU YG, et al. Rapid analysis of copy number of foreign genes in transgenic maize by micro-drop digital PCR [J]. Biotechnol Prog, 2016, 6 (4): 288
[17] 姜羽,胡佳莹,杨立桃.利用微滴数字PCR分析转基因生物外源基因拷贝数[J]. 农业生物技术学报,2014,22(10):1298
JIANG Y, H JY, YANG LT. Analysis of foreign gene copy number of transgenic organisms by micro-drop digital PCR [J]. J Agric Biotech, 2014, 22 (10): 1298
