The Role of ddPCR in Developing Safe and Effective AAV-Based Gene Therapies

The Promise and Challenges of Gene Therapy

With their capacity to yield semi-permanent solutions to diseases previously thought to be intractable, adeno-associated virus (AAV)–mediated gene therapies may soon revolutionize medicine. In the U.S., the first gene therapy product was approved by the Food and Drug Administration (FDA) in 2017 (U.S. Food & Drug Administration, 2017), helping return sight to patients affected by a type of inherited retinal disease. Countless other diseases, from hemophilia B to spinal muscular atrophy, could soon follow suit (U.S National Library of Medicine, 2021).

Behind the scenes, however, the process of generating AAV vectors for gene therapy is imperfect. Viral particles are produced in cells, then purified, making it hard to control the concentration of the virus in the final product. The process is also susceptible to contamination by host cell DNA (which can be oncogenic), mycoplasma (which can cause respiratory illnesses), and other contaminants (Razin 1996, Hebben 2008). Viral titer must be measured accurately and precisely to ensure patients receive the correct dose. If the titer is too low, the treatment may not be effective. If it’s too high, it can cause lethal complications, such as cytokine storm.

Solutions for AAV-Based Gene Therapy Manufacturing

To mitigate these risks, developers of AAV-based gene therapies need a sensitive and accurate tool for measuring viral titers and detecting contaminants in finished gene therapy products.

While qPCR remains the gold standard tool for many applications, including the measurement of nucleic acids, it lacks the high sensitivity and precision needed to accurately gauge AAV viral titers. This is due, in part, to the fact that qPCR must be analyzed via a standard curve, which can cause its results to vary by up to a factor of two (DeMaio 2020). Also, qPCR measurements are sensitive to the inhibitory effects of secondary structures in DNA reference standards, which can lead to an overestimation of viral titer (Furuta-Hanawa et al. 2019).

ddPCR technology is an appealing alternative to qPCR. Unlike qPCR, ddPCR can provide an absolute count of nucleic acids, enabling the precise quantification of AAV vectors, bacterial contaminants, and residual host cell DNA (Dobnik et al. 2019). No reference standard is required, improving precision and removing a source of variation. ddPCR is also less sensitive to contaminants that affect amplification, including those present in solutions used during the development of AAV-mediated gene therapies.

In one direct comparison of qPCR and ddPCR technology, ddPCR was up to four times more sensitive than qPCR in the absolute quantification of single-stranded AAV vector genomes (Lock 2020). This level of accuracy and precision is critical for gene therapy development, as it makes it easier to assess the potency of a treatment and determine the correct dose a patient should receive.

With standard drugs, manufacturing is fairly predictable. Using a bit of stoichiometry and mathematics, a conventional drug developer can predict quantities of active drug versus contaminants in a dose, as well as the medicine’s potency. But virus-mediated gene therapies represent a new kind of biological product where not every aspect of manufacturing can be controlled, leading to greater uncertainty in the molecular makeup of the final therapeutic batch. As gene therapies become more common, the need to quantify viral particles and contaminants with tools like ddPCR technology will only increase.

Visit our Cell and Gene Therapy Resources page to learn more about how ddPCR technology offers the accuracy required to support many aspects of quality control for AAV-based gene therapy development.

References