Column on Quality Evaluation Techniques and Methods for Gene Therapy Products(2)

Exploration and application of multiplex dPCR in rAAV genome integrity evaluation*

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  • National Institutes of Food and Drug Control, Beijing 100050, China

Revised date: 2023-03-15

  Online published: 2024-06-21

Abstract

Objective: To explore the feasibility of multiplex PCR technique in recombinant adeno-associated virus(rAAV) genome integrity evaluation. Methods: A chip dPCR system was used to establish a dual PCR method targeting ITR region and gene of interest (or the 3’untranslated region) of rAAV genome. By proper dilution, a theoretical single copy distribution of template DNA was achieved in the PCR chip wells (the parentage of positive wells was less than 20%), the single positive wells represented the incomplete fragment, and the double positive wells represented the complete sequence between two sets of primer pairs. The integrity of rAAV genome was evaluated by testing the ratio of double positive wells. Different sample pretreatment methods (virus genomic DNA, virus particles, DNase Ⅰ treatment) were conducted to compare the test results. Results: The copy number measured by duplex PCR was basically consistent with that of single PCR, showing no obvious interference. Within a certain range, the measured copy number of samples was linearly correlated with the amount of templates, but the proportions of single positive and double positve was basically the same. Compared with untreated viral particles, the measured copy number of the extracted viral genome DNA was reduced, but the double positive rate was higher. The measured copy number of the virus samples treated with DNaseⅠ was significantly reduced, and the proportions of single positive and double positive were basically consistent with those of the untreated viral samples. Conclusion: Multiplex dPCR technology could be used to evaluate the genome integrity of rAAV products, showing a good application prospect.

Cite this article

YU Lei, WANG Guang-yu, FAN Wen-chao, SHI Xin-chang, ZHOU Yong . Exploration and application of multiplex dPCR in rAAV genome integrity evaluation*[J]. Chinese Journal of Pharmaceutical Analysis, 2023 , 43(12) : 1997 -2003 . DOI: 10.16155/j.0254-1793.2023.12.01

References

[1] 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
[2] 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
[3] NASO MF, TOMKOWICZ B, PERRY WL, et al. Adeno-associated virus (AAV) as a vector for gene therapy[J]. BioDrugs, 2017,31(4):317
[4] MENDELL JR, AL-ZAIDY SA, RODINO-KLAPAC LR, et al. Current clinical applications of in vivo gene therapy with AAVs[J]. Mol Ther, 2021,29(2):464
[5] RODRIGUEZ-MARQUEZ E, MEUMANN N, BUNING H. Adeno-associated virus (AAV) capsid engineering in liver-directed gene therapy[J]. Expert Opin Biol Ther, 2021,21(6):749
[6] KONDRATOV O, MARSIC D, CROSSON SM, et al. Direct head-to-head evaluation of recombinant adeno-associated viral vectors manufactured in human versus insect cells[J]. Mol Ther, 2017,25(12):2661
[7] SANDRO Q, RELIZANI K, BENCHAOUIR R. AAV production using baculovirus expression vector system[J]. Methods Mol Biol, 2019,1937: 91
[8] GUAN JS, CHEN K, SI Y, et al. Process improvement of adeno-associated virus (AAV) production[J]. Front Chem Eng, 2022, 4: 830421
[9] HAJBA L, GUTTMAN A. Recent advances in the analysis full/empty capsid ratio and genome integrity of adeno-associated virus (AAV) gene delivery vectors[J]. Curr Mol Med, 2020,20(10):806
[10] TUSTIAN AD, BAK H. Assessment of quality attributes for adeno-associated viral vectors[J]. Biotechnol Bioeng, 2021,118(11):4186
[11] GIMPEL AL, KATSIKIS G, SHA S, et al. Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies[J]. Mol Ther Methods Clin Dev, 2021,20: 740
[12] TRAN NT, HEINER C, WEBER K, et al. AAV-Genome population sequencing of vectors packaging crispr components reveals design-influenced heterogeneity[J]. Mol Ther Methods Clin Dev, 2020,18: 639
[13] TAI PWL, XIE J, FONG K, et al. Adeno-associated virus genome population sequencing achieves full vector genome resolution and reveals human-vector Chimeras[J]. Mol Ther Methods Clin Dev, 2018,9: 130
[14] NAMKUNG S, TRAN NT, MANOKARAN S, et al. Direct ITR-to-ITR nanopore sequencing of AAV vector genomes[J]. Hum Gene Ther, 2022,33(21-22):1187
[15] TRAN NT, LECOMTE E, SALEUN S, et al. Human and insect cell-produced recombinant adeno-associated viruses show differences in genome heterogeneity[J]. Hum Gene Ther, 2022,33(7-8):371
[16] PENAUD-BUDLOO M, LECOMTE E, GUY-DUCHE A, et al. Accurate identification and quantification of dna species by next-generation sequencing in adeno-associated viral vectors produced in insect cells[J]. Hum Gene Ther Methods, 2017,28(3):148
[17] EMILIE L, BENOIT T, BENJAMIN C, et al. Advanced characterization of dna molecules in raav vector preparations by single-stranded virus next-generation Sequencing[J]. Mol Ther Nucleic Acids, 2015, (10):e260
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