Recombinant adeno-associated virus (rAAV) vectors have been widely used for in vivo gene therapy since Luxturna™ and Zolgensma™ received FDA approval for use as commercial products in 2017 and 2019, respectively.
rAAV, however, is designed to not replicate, so rAAV vector characterization would typically require a purposely engineered cell line and a helper virus to support in vitro viral replication.
Up to 60% assay-to-assay variability has been observed in infectious titer analysis of rAAV vectors using such artificial experimental systems. Genome titer of rAAV vectors (over infectious titer) has been commonly used for clinical dosing, since accuracy and reproducibility of rAAV titer are critical. Historically, real-time PCR (qPCR) technology has been used to measure rAAV vector genome titer.
Genome titer quantification by qPCR relies heavily on the standard curve derived from a dilution series of a known quantity of plasmid DNA, which typically is created and quantified by an individual laboratory without any reference standard. Using qPCR, approximately 15–20% variability has been observed batch-to-batch and/or within the same batch over time.
A Platform Approach for Analytical Methods
to Support Adeno-Associated Virus (AAV) Gene Therapy Products
This webinar examines data from in-house development and generic validation studies and discusses individualizing platform analytical methods based on a specific AAV gene therapy product.
Absolute Quantification, Greater Precision
Droplet Digital PCR (ddPCR) technology has been gaining acceptance for measuring rAAV vector genome titer by both the gene therapy industry and regulatory agencies. ddPCR technology uses water-oil emulsion droplet technology to fractionate a sample into 20,000 droplets, with target-specific PCR amplification occurring in each individual droplet. Following PCR, each droplet is analyzed to determine the presence (positive droplet) or the absence (negative droplet) of the intended target.
A good separation from positive vs. negative wells with minimum rain achieved by the optimized primer/probe design in ddPCR assay.
The droplet data is then used to calculate the absolute target quantity by Poisson statistics in the original sample. Absolute quantification of rAAV vector genome titer by ddPCR eliminates the need for the dilution series of standard, which is a major source of qPCR assay variability.
In general, ddPCR also has a higher tolerance of matrix interference, since low-efficiency PCR reactions in any droplets due to inhibition are still counted as positive droplets. Taken together, the unparalleled degree of precision demonstrated by ddPCR in the absolute quantification of the intended target has made this technology a highly suitable method for rAAV vector genome titration to support clinical dosing.
Our Method Development Group has developed a ddPCR-based rAAV vector genome titration assay, which is now available to support your diverse rAAV vector program.
If you are developing an rAAV product and need to determine its genome titer, our platform assay can be customized to your particular transgene cassette.