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Column on Quality Evaluation Techniques and Methods for Gene Therapy Products（2）
Editor: Shief editor of invited column： SHI Xin-chang

    Gene therapy refers to the method of treating diseases by introducing nucleic acid fragments into cells in an appropriate manner, allowing the genetic information of nucleic acids to function accordingly. Gene therapy products are tools for implementing this method.

    Since the 1990s, gene therapy has developed rapidly. In this century, a number of gene therapy products at home and abroad have been approved for marketing. Among them, seven varieties of recombinant adeno-associated virus (rAAV) vector gene therapy products have been approved for marketing worldwide. In the past three years, 26 rAAV products have been approved for clinical trials at home, with indications including ophthalmic diseases, blood system diseases, nervous system diseases and infectious diseases, and serotypes involving 2type, 5type, 8type, 9type and 10type.

    As more and more gene therapy products enter clinical practice, potential safety risks are gradually being exposed. Therefore, regulatory authorities in various countries are accelerating the development of relevant regulations and technical guidelines to guide the research and evaluation of gene therapy products. The 2020 edition Pharmacopoeia of the People's Republic of China, Parts Ⅲ, “General Introduction to Human Gene Therapy Products” and the relevant guiding principles for rAAV gene therapy products issued by the Center for Drug Evaluation (CDE) of the National Medical Products Administration, which have put forward principle requirements for quality control. However, given that rAAV is a “live” form of virus, its higher requirements for production and quality control and the current testing method continuously improving are still required, including the durability of the method and the repeatability of the results. Therefore, in order to promote the early application of new concepts, technologies, tools, and standards in the quality control of gene therapy products, a “Gene Therapy Product Quality Evaluation Technology and Methods Column” has been specially established to showcase relevant research progress of quality analysis technology. The column includes 9 articles published in 2 journals. Five papers were published in 2023(11), the others published in column(2) of the jounal with following key contents： 1.Exploration and application of multiple digital PCR in rAAV genome integrity evaluation, explores the quantitative methods for the complete genome of rAAV products. 2.Detection of plasmid DNA residues in rAAV products by digital PCR technology, explores a method of characterizing residue by plasmid DNA copy number. 3.Detection of replication-competent AAV(rcAAV) in rAAV2-ND4 injection by qPCR, explores a method for detecting rcAAV through 2-round culture and qPCR. 4.Sequence analysis of amino acid residues in adeno-associated virus capsid protein of different serotypes, explores the principle of serotypes changes in rAAV products.

    It’s hoped to use the columns to attract the attention of relevant enterprises and research institutions to the quality of rAAV products, continuously to improve the understanding of practitioners in the dimension of rAAV product quality control, to improve inspection and testing capabilities, and to explore and establish corresponding experimental plans to promote the resolution of difficult problems, thereby promoting the construction of China's rAAV gene therapy product quality control system and the improvement of technical specifications, and accelerating the realization of the grand goal of becoming a pharmaceutical powerhouse.

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  • Column on Quality Evaluation Techniques and Methods for Gene Therapy Products（2）
    Exploration and application of multiplex dPCR in rAAV genome integrity evaluation*
    YU Lei, WANG Guang-yu, FAN Wen-chao, SHI Xin-chang, ZHOU Yong
    Chinese Journal of Pharmaceutical Analysis. 2023, 43(12): 1997-2003. https://doi.org/10.16155/j.0254-1793.2023.12.01
    Abstract (25) PDF (8)   Knowledge map   Save
    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.
  • Column on Quality Evaluation Techniques and Methods for Gene Therapy Products（2）
    Detection of plasmid DNA residues in rAAV products by digital PCR technology *
    FAN Wen-chao, YU Lei, AN Yi-fang, WANG Guang-yu, SHI Xin-chang, ZHOU Yong
    Chinese Journal of Pharmaceutical Analysis. 2023, 43(12): 2004-2009. https://doi.org/10.16155/j.0254-1793.2023.12.02
    Abstract (14) PDF (8)   Knowledge map   Save
    Objective: To detect plasmid DNA residues in recombinant adeno-associated virus (rAAV) products by digital PCR (dPCR). Methods: Primer probes were designed for four different segments of the plasmid resistance gene KanR sequence, and singleplex and duplex dPCR were established to detect KanR residues. In duplex dPCR, double positive wells represent the sequence integrity between two sets of primer pairs, and the distribution of KanR sequences was assessed by testing the proportion of single and double positive wells. The presence of incorrectly packaged plasmid DNA within the viral capsid was evaluated by digesting free plasmid DNA with DNase Ⅰ. Results: The KanR residues measured in four groups by single dPCR were basically consistent (1.054×108-1.467×108) copies·μL-1. There was no significant difference between the measured results of duplex dPCR and singleplex dPCR in each channel. It showed good linearity (r＞0.999) and repeatability (RSD<20%) in 6-6 000 copies·μL-1 concentration range for the established duplex dPCR. The proportion of double positive wells in the dual PCR of 1/2, 2/3, and 3/43 combinations was basically consistent, indicating that the breakage of the KanR gene may tend to be more random. The results before and after DNase Ⅰ treatment suggested that the proportion of large fragments of plasmid DNA (1/4 double positive) that are wrongly packaged was higher than that of free plasmid DNA. Conclusion: The dPCR technology can be used to detect plasmid DNA residues in rAAV products, and multiple dPCR can also evaluate the size and distribution of plasmid DNA fragments.
  • Column on Quality Evaluation Techniques and Methods for Gene Therapy Products（2）
    Detection of replication-competent AAV2(rcAAV2) in rAAV2-ND4 injection by qPCR*
    QIN Xi, CHEN Long, YANG Jing-qing, LI Xiang, ZHOU Feng, SHI Xin-chang, BI Hua, WANG Guang-yu, ZHU Liu-qiang, AN Yi-fang, PEI De-ning, ZHOU Yong
    Chinese Journal of Pharmaceutical Analysis. 2023, 43(12): 2010-2016. https://doi.org/10.16155/j.0254-1793.2023.12.03
    Abstract (9) PDF (6)   Knowledge map   Save
    Objective: To develop and validate a qPCR method for the determination of replication-competent AAV2(rcAAV2) in rAAV2-ND4. Methods: HEK293 cells (6.0×105 cells·mL-1, 1 mL per well, inoculated into a 12 well plate for 24 h at 37 ℃ and 5% carbon dioxide) were infected with rAAV2 ND4 sample (3.0×1010 vg·mL-1, with 100 μL per well) and wtAd5 (6.0×109 TCID50·mL-1, with 10 μL per well) simultaneously. After 2 h of infection, the infected medium was replaced with cell culture medium (DMEM containing 10% FBS, 1 mL per well), and then the infected cells were cultured at 37 ℃ and 5% carbon dioxide for another 48 h. The cells were collected and treated with cell lysate solution. The cell lysate was then subjected to the above steps for infection and culture. The cells from the second culture were collected and incubated with lysate at 56 ℃ for 30 min, followed by incubation at 90 ℃ for 10 min. The processed cell lysates were detected for rcAAV2 using qPCR method, the qPCR reaction system included 2×T5 Fast qPCR Mix (Probe) 10 μL, primer mixture 0.1 μL, probe 1 μL, 50×ROX Reference Dye I 0.4 μL, EASY Dilution 3.5 μL, treated cell lysate 5 μL, and qPCR amplification program was set as bellow: 95 ℃, 10 min; 40 cycles of [95 ℃, 15 s; 60 ℃, 1 min]. Then the sensitivity, accuracy, precision, and specificity of the method were verified. Results: The sensitivity of the established method for detecting rcAAV2 reached less than 1 TCID50 rcAAV per 108 virus genomes; the recovery rate of qPCR method ranged from 77.6% to 91.2%. The same results in three experiments per person (two people) was obtained, and the method specificity was good. Conclusion: The established qPCR method after two rounds of infection amplification can be applied to the detection of rcAAV2 in rAAV2 ND4 samples.
  • Column on Quality Evaluation Techniques and Methods for Gene Therapy Products（2）
    Sequence analysis of amino acid residues in adeno-associated virus capsid protein of different serotypes*
    HOU Zong-wen, QIN Xi, TAO Lei, SHI Xin-chang, ZHANG Yi-xuan
    Chinese Journal of Pharmaceutical Analysis. 2023, 43(12): 2017-2024. https://doi.org/10.16155/j.0254-1793.2023.12.04
    Abstract (14) PDF (6)   Knowledge map   Save
    Objective: To analyze the amino acid residue sequences of adeno-associated virus capsid protein (VP1) of different serotypes, and establish the similarity comparison method and database, and to predict the serotypes that VP1 may belong to after amino acid residue sequence mutation. Methods: The amino acid residues of VP1 of adeno-associated virus of different serotypes were analyzed to find out a common sequence, and the sequences were aligned. The hydrophobic amino acid residues, hydrophilic amino acid residues and acid/base amino acid residues were assigned separately. The assignment parameters included side chain length, group number and dissociation/binding strength. Then the similarity was calculated by correlation coefficient method and difference method. The global charge was defined to determine the boundary value, and determine whether the change of charge will cause a significant change in the electric field around the protein. Finally, considering the relationship of the four, the similarity with the known serological sequences was given. The protein amino acid sequences of other known serotypes were verified. Results: Basically, different serotypes could be distinguished, and different subtypes of the same serotype have a certain tolerance. Conclusion: The relationship between the amino acid residues sequence of adeno-associated VP1 and its serotype was discussed, hoping to provide support for the research and development of recombinant adeno-associated virus as a vector for gene therapy.
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