ABSTRACT
@#ObjectiveTo develop a national standard for genomic titer determination of recombinant type 5 adeno-associated virus(rAAV5).MethodsThe rAAV5-GFP stock solution prepared by the three-plasmid system was identified and verified for the appearance,pH,sterility,genomic titer,purity and infection titer according to the relevant requirements of Chinese Pharmacopoeia(Volume Ⅲ,2020 edition),which was diluted and subpackaged to prepare candidate standards according to the results;The stability of candidate standards was investigated by thermal acceleration test;Three laboratories were organized to collaboratively calibrate the candidate standards using droplet digital PCR(ddPCR).ResultsAll the detection indexes of the candidate standard and the stock solution met the relevant requirements;The genomic titer showed no significant decrease at 25,4,-20,-40,-80 ℃ for 1,3,4,6 months;Through collaborative calibration by three laboratories,the candidate standard was assigned a value of 2. 56 × 10(12)copies/mL,and the 95% confidence interval was 2. 48 ×10(12)copies/mL,and the 95% confidence interval was 2. 48 ×10(12)copies/mL ～ 2. 64 × 10(12)copies/mL ～ 2. 64 × 10(12)copies/mL.ConclusionThe developed national standard for the determination of rAAV5 genomic titer had good stability and might be used for the quality evaluation of rAAV5 related products.
ABSTRACT
OBJECTIVES: To develop a method for reliable quantification of viral vectors, which is necessary for determining the optimal dose of vector particles in clinical trials to obtain the desired effects without severe unwanted immune responses. RESULTS: A significant level of vector plasmid remained in retroviral and lentiviral vector samples, which led to overestimation of viral titers when using the conventional RT-qPCR-based genomic titration method. To address this problem, we developed a new method in which the residual plasmid was quantified by an additional RT-qPCR step, and standard molecules and primer sets were optimized. The obtained counts were then used to correct the conventionally measured genomic titers of viral samples. While the conventional method produced significantly higher genomic titers for mutant retroviral vectors than for wild-type vectors, our method produced slightly higher or equivalent titers, corresponding with the general idea that mutation of viral components mostly results in reduced or, at best, retained titers. CONCLUSION: Subtraction of the number of residual vector plasmid molecules from the conventionally measured genomic titer can yield reliable quantification of retroviral and lentiviral vector samples, a prerequisite to advancing the safety of gene therapy applications.
