Real-Time qPCR for rAAV Genome Copy Number Titration

Recombinant adeno-associated virus (rAAV) vectors have many advantages for gene therapeutic applications compared with other vector systems. Despite the rapid progress in this area, the coordination of vector analysis and dose units remains a limitation to commercialization. AAV reference standard materials help ensure product safety by controlling the consistency of analysis used to characterize rAAV inventory. The most widely used vector dose unit is based on the encapsulated vector genome. Quantitative polymerase chain reaction (qPCR) is the most commonly used method for the titer vector genome. At present, Creative Biolabs has mastered this technology and applied it skillfully to the titration determination of AAV genome copy number.

Real-Time qPCR to Titrate rAAV Genome Copy Number

Gene and cell therapy use a variety of viral vectors including rAAV for gene transfer. At present, the application of viral vectors in basic and clinical research has attracted more and more attention, which has prompted people to strive to improve the quality and yield of rAAV production. Standard vector production workflow requires genomic titration of purified vectors at the end of production to assess yield. qPCR plays a key role in many analytical methods for characterizing and quantifying these viral vectors during production, release and stability testing. So far, traditional real-time qPCR analysis has been used to analyze the distribution of rAAV vectors in human trials or animal models to determine the level of gene delivery in target tissues.

rAAV quantification by qPCR.Figure 1. rAAV quantification by qPCR.

Method for rAAV Genome Copy Number Titration

qPCR is the most commonly used method to titrate the vector genome. The general steps for titrating rAAV genome include:

  • The vector was pretreated with DNA enzyme I to eliminate unpackaged AAV DNA or contaminated plasmid DNA;
  • After heat treatment, the virus genome packaged with endonuclease was released by protease K;
  • Real-time PCR quantification was performed using a set of criteria containing known copies.

In order to achieve high throughput titration, primers and probes used in real-time PCR are usually designed to target common elements in most rAAV genomes, such as promoters and poly-A signals. With the reaction proceeding, the amplified products were detected in real-time by the fluorescence method. We have proved that this method is accurate and extensible, and can be applied to the vector genome titration of single-stranded and self-complementary AAV genomes. This strategy significantly reduces the time of PCR, control and turnaround. Therefore, this qPCR method has great significance in reducing time and cost burden, quality control evaluation in the process, batch/lot monitoring in large-scale preparation and good production practice for rAAV vector production.

Creative Biolabs provides high-quality titration services in the manufacture of rAAV. For more details about our virus service, please contact us.

For research use only. Not intended for any clinical use.