重组9型腺相关病毒基因组滴度测定方法的改进和初步验证

[1] 肖桂清,杨会勇,刁勇.重组腺相关病毒质量控制的qPCR技术研究进展[J].华侨大学学报(自然科学版),2014,35(2):191 XIAO GQ,YANG HY,DIAO Y.Advances in real-time quantitative PCR technology for quality control of recombinant adeno-associated virus[J].J Huaqiao Univ(Nat Sci),2014,35(2):191
[2] 杜梦潭,刘兴健,胡小元,等. 腺相关病毒的生产方式及其在基因治疗中的应用[J]. 生物技术进展,2019,9(4):326 DU MT,LIU XJ,HU XY,et al.The production method of adeno-associated virus and its application in gene therapy[J].Curr Biotechnol,2019,9(4):326
[3] 刁勇,王启钊,吕颖慧,等.重组腺相关病毒基因药物的病毒滴度定量测定[J].中国新药与临床杂志,2010,29(10):728 DIAO Yo,WANG QZ,Lü YH,et al.Biological assay of recombinant adeno-associated virus gene medicine[J].Chin J New Drugs Clin Rem,2010,29(10):728
[4] WANG D,TAI PWL,GAO G.Adeno-associated virus vector as a platform for gene therapy delivery[J].Nat Rev Drug Discov,2019,18(5):358
[5] 蒙青林,张彬彬,张春. 测定重组腺相关病毒基因组滴度的qPCR新方法[J].生物工程学报,2013,29(2):235 MENG QL,ZHANG BB,ZHANG C.Novel qPCR strategy for quantification of recombinant adeno-associated virus serotype 2 vector genome-titer[J].Chin J Biotechnol,2013,29(2):235
[6] WAGNER A,R?HRS V,KEDZIERSKI R,et al.A novel method for the quantification of adeno-associated virus vectors for RNA interference applications using quantitative polymerase chain reaction and purified genomic adeno-associated virus DNA as a standard[J].Hum Gene Ther Methods,2013,24(6):355
[7] 石亮,刘云波.腺相关病毒的特性及应用进展[J].医学综述,2016,22(11):2088 SHI L,LIU YB. The characteristics and applications of adeno-associated virus[J].Med Recapit,2016,22(11):2088
[8] AURNHAMMER C,HAASE M,MUETHER N,et al.Universal real-timePCR for the detection and quantification of adeno-associated virus sero-type 2-derived inverted terminal repeat sequences[J].Hum Gene Ther Methods,2012,23(1):18
[9] BP 8.0[S].2013:609
[10] 于雷,李永红,秦玺,等.Q-PCR法检测腺病毒基因治疗产品中的复制型腺病毒[J].生物技术通讯,2017,28(3):352 YU L,LI YH,QIN X,et al.Detection of replication-competent adenoviruses in adenoviral gene therapy products by Q-PCR[J].Lett Biotechnol,2017,28(3):352
[11] D'COSTA S,BLOUIN V,BROUCQUE F,et al.Practical utilization of recombinant AAV vector reference standards:focus on vector genomes titrationby free ITR qPCR[J].Mol Ther Methods Clin Dev,2016,5:16019

Objective: To optimize and preliminarily validate a method for genome titer determination of recombinant adeno-associated virus type 9(rAAV-9) with the aim of improving the precision of test results.Methods: The method of virus dissociation was optimized by comparing the effect of SDS and EDTA,as well as SDS at different concentrations;an appropriate reference standard was selected by comparing the test results using plasmid or virus as the reference standard;an appropriate statistical method was selected by comparing the results of parallel line method and traditional single point method.Results: The results showed that the dissociation effect of SDS was significantly better than that of EDTA,and the effect of SDS was the best when its final concentration was 0.1%.The results of assay using virus as reference standard and parallel line method as statistical method showed the lowest variability (RSD of 13.7%),which was better than the assay using plasmid as reference standard (RSD of 35.9%) and single point method as statistical method (RSD of 23.1%).Conclusion: The optimized method is superior to the traditional method for genome titer determination of recombinant AAV and can be applied to the quality control of recombinant AAV-9 gene therapy products.

[1] 肖桂清,杨会勇,刁勇.重组腺相关病毒质量控制的qPCR技术研究进展[J].华侨大学学报(自然科学版),2014,35(2):191 XIAO GQ,YANG HY,DIAO Y.Advances in real-time quantitative PCR technology for quality control of recombinant adeno-associated virus[J].J Huaqiao Univ(Nat Sci),2014,35(2):191
[2] 杜梦潭,刘兴健,胡小元,等. 腺相关病毒的生产方式及其在基因治疗中的应用[J]. 生物技术进展,2019,9(4):326 DU MT,LIU XJ,HU XY,et al.The production method of adeno-associated virus and its application in gene therapy[J].Curr Biotechnol,2019,9(4):326
[3] 刁勇,王启钊,吕颖慧,等.重组腺相关病毒基因药物的病毒滴度定量测定[J].中国新药与临床杂志,2010,29(10):728 DIAO Yo,WANG QZ,Lü YH,et al.Biological assay of recombinant adeno-associated virus gene medicine[J].Chin J New Drugs Clin Rem,2010,29(10):728
[4] WANG D,TAI PWL,GAO G.Adeno-associated virus vector as a platform for gene therapy delivery[J].Nat Rev Drug Discov,2019,18(5):358
[5] 蒙青林,张彬彬,张春. 测定重组腺相关病毒基因组滴度的qPCR新方法[J].生物工程学报,2013,29(2):235 MENG QL,ZHANG BB,ZHANG C.Novel qPCR strategy for quantification of recombinant adeno-associated virus serotype 2 vector genome-titer[J].Chin J Biotechnol,2013,29(2):235
[6] WAGNER A,RÖHRS V,KEDZIERSKI R,et al.A novel method for the quantification of adeno-associated virus vectors for RNA interference applications using quantitative polymerase chain reaction and purified genomic adeno-associated virus DNA as a standard[J].Hum Gene Ther Methods,2013,24(6):355
[7] 石亮,刘云波.腺相关病毒的特性及应用进展[J].医学综述,2016,22(11):2088 SHI L,LIU YB. The characteristics and applications of adeno-associated virus[J].Med Recapit,2016,22(11):2088
[8] AURNHAMMER C,HAASE M,MUETHER N,et al.Universal real-timePCR for the detection and quantification of adeno-associated virus sero-type 2-derived inverted terminal repeat sequences[J].Hum Gene Ther Methods,2012,23(1):18
[9] BP 8.0[S].2013:609
[10] 于雷,李永红,秦玺,等.Q-PCR法检测腺病毒基因治疗产品中的复制型腺病毒[J].生物技术通讯,2017,28(3):352 YU L,LI YH,QIN X,et al.Detection of replication-competent adenoviruses in adenoviral gene therapy products by Q-PCR[J].Lett Biotechnol,2017,28(3):352
[11] D'COSTA S,BLOUIN V,BROUCQUE F,et al.Practical utilization of recombinant AAV vector reference standards:focus on vector genomes titrationby free ITR qPCR[J].Mol Ther Methods Clin Dev,2016,5:16019