For gene therapy AAV titer determination, not all methods are created equal

The advent of gene therapy has unlocked new possibilities for treating diseases previously regarded as intractable. However, producing adeno-associated virus (AAV)-mediated gene therapy products presents challenges and risks unique to this therapeutic platform. To create an AAV-mediated gene therapy product, viral particles are produced in cultured cells and then purified. But safe and effective dosing of a gene therapy product depends on accurately determining the viral titer, and the stakes are high. An insufficient concentration may fail to be effective, but an excessive concentration can produce potentially lethal complications, such as cytokine release syndrome (CRS). To safeguard against these consequences and ensure accurate viral titer determination, manufacturers rely on highly precise and sensitive quantification methods.

A revolution in analytical capabilities

Although qPCR remains the gold standard for many nucleic acid measurement applications, it falls short in the high precision and sensitivity needed to quantify AAV titers accurately. Digital PCR (dPCR), including Droplet Digital^TM PCR (ddPCR^TM), has emerged as the gold standard to titer vector genome, a better alternative that overcomes many of the limitations presented by qPCR. Because ddPCR technology works by partitioning a sample into thousands of individual PCR reactions, which are then analyzed on a positive/negative binary, it provides absolute quantification of the nucleic acid present in the sample. In contrast, qPCR requires the use of a standard curve, which can cause results to vary considerably. Eliminating the need for a reference standard curve in dPCR thus removes a source of variation and improves overall precision. In one study directly comparing the two methods, ddPCR technology was up to four times more sensitive than qPCR in quantifying single-stranded AAV genomes. When determining a safe and effective dose for AAV-mediated gene therapy, this level of sensitivity can make a world of difference.

Leveraging the full power of digital PCR

While dPCR is an overall superior method for quantification of viral titer in gene therapy production, not all data are created equal. The success of dPCR analysis hinges upon accurately discerning “positive” partitions that contain the target sequence from “negative” partitions that do not. High-quality dPCR data should thus have a clearly defined threshold with tight and consistent amplitudes for positive and negative partitions. If partition amplitudes vary or exhibit “jitter,” then this differentiation may be jeopardized. Irregular amplitudes can also make threshold setting difficult, especially if partitions are poorly separated. In these cases, slight variations in the threshold can drastically affect quantification and decrease the accuracy of the data.

Understanding factors impacting data quality can ensure that dPCR is used to its fullest potential. Above all, quality results are impossible without quality tools, so choosing a robust dPCR platform that consistently delivers partitions of a defined size is essential. Additionally, a dPCR instrument should have a stable optical bench and high-quality optics to minimize noise and maximize sensitivity. Validated assays are also key in ensuring dPCR data quality. If an assay lacks target sequence specificity, results may lack clarity or include random, false positive data points. A high-quality dPCR assay will have high amplification efficiency and a strong signal for the target sequence. Factors outside of the tools can also impact dPCR data quality, so users must avoid cross-contamination and use instruments and assays as directed. Selecting a dPCR platform with user-friendly data analysis software can also ensure confidence and clarity when assessing results.

Accelerating safe and effective gene therapies

AAV-mediated gene therapy products can provide new hope to patients with serious health conditions, but this cannot be accomplished without reliable methods to quantify viral titer for dosing accurately. dPCR has offered developers unprecedented accuracy, sensitivity, and precision, moving the industry forward. However, not all dPCR tools are created equal—knowing the factors that can impact data quality can help users choose instruments and assays that ensure safe and effective gene therapies, one partition at a time.

Learn more about leveraging dPCR for viral vector characterization and optimizing dPCR data