Multiplex digital PCR to detect the titer of recombinant adeno-associated virus (rAAV) viral genome copy number

【Introduction】

Aperbio and Xining (Jinghan) Bio have joined hands to carry out in-depth cooperation on multiplex digital PCR detection of the titer of the recombinant adeno-associated virus (rAAV) viral genome copy number. This powerful combination uses Naica Crystal digital PCR technology (cdPCR) to optimize the genome copy number quantification process of recombinant adeno-associated viruses rAAV2 and rAAV8.

【Overview】

Recombinant adeno-associated virus (rAAV) is a key viral vector for gene therapy. The main challenges in the development of this viral vector come from the upstream and downstream process optimization and standardization of detection schemes in large-scale production. Among them, the accurate titration of the recombinant adeno-associated virus (rAAV) genome copy number is directly related to the correct dosage in preclinical and clinical environments. Establishing a standard, accurate and reproducible method for quantifying the recombinant adeno-associated virus (rAAV) genome copy number is an important part of process development and optimization in gene therapy products. It has been reported that the use of real-time fluorescence quantitative PCR (qPCR) technology for titration detection of recombinant adeno-associated virus (rAAV) genome copy number has problems such as complex standard establishment and poor quantitative repeatability. Digital PCR (dPCR) technology, as a new PCR technology, can directly quantify the recombinant adeno-associated virus (rAAV) genome copy number without relying on standards and standard curves. It has a high tolerance to inhibitors and is particularly suitable for quantitative detection of unpurified virus lysis samples.

Fig1. Production of recombinant adeno-associated virus (rAAV)

(1) When the ITR-flanked expression vector plasmid carrying the target gene sequence was co-transfected with

(3) the adenovirus helper plasmid into HEK-293 cells, the production efficiency of the AAV vector was consistent with that when adenovirus was used for infection.

【 Experimental purpose 】

We used Naica Crystal digital PCR technology (cdPCR) to optimize the method for simultaneous copy number quantification of three different gene sites in the recombinant adeno-associated virus (rAAV) plasmid vector and viral genome: ITR (CY5 marker), EGFP (GOI, HEX marker) and transcription-related regulatory element WPRE (FAM marker). In the data analysis, the conditions of the pretreatment schemes of different virus samples were described and compared. It was found that the linearization of the ITR hairpin structure and the cleavage conditions of the capsid protein in the pretreatment scheme of the virus sample were particularly important for the accuracy of the quantification of the viral genome copy number, providing a basis for the establishment of a standard detection method for the digital PCR method in the quality control system of gene therapy viral vectors.

【 Quantitative detection scheme for rAAV viral genome copy number titer 】