(M1330-11-60) The Effects of Interfering Viral Particles on the Probability of Detecting Replication-Competent Adeno-Associated Virus (rcAAV) in a Replication-Based Cell Assay

Purpose: Adeno-associated viruses (AAV) are a popular and versatile platform for gene therapy. Recombinant AAV (rAAV) created for gene therapy are replication incompetent as they lack the Rep and Cap genes required for replication. However, rare recombination events during manufacturing can result in replication-competent AAV (rcAAV) in the viral drug product, which may pose a risk to patients. FDA and ICH guidelines state that viral drugs must be tested for rcAAV prior to release. Testing can be either a PCR-only assay or a replication-based assay. PCR-only assays test for nucleic acid sequences suggestive of rcAAV. Replication-based assays work by allowing rcAAV to replicate over serial infections in cells. While PCR-only assays for rcAAV are faster, they cannot distinguish between nucleic acid sequences and infectious replicating viral particles. Replication-based assays detect viral replication, but due to the nature of viral infection at low concentrations of virus, this format has probabilistic outcomes. Detecting rcAAV in a viral drug sample means it is necessary to detect low concentrations of rcAAV in relatively high concentrations of non-replicating rAAV (non-rcAAV) particles. Testing a known replicating AAV like wild-type AAV (wtAAV) by itself acts as a ‘positive control’, demonstrating that rcAAV can replicate under ideal conditions. Testing wtAAV in the presence of a higher concentration of non-rcAAV is an ‘interference control’, mimicking the presence of low concentrations of rcAAV in a non-rcAAV drug sample. The lower the concentration of rcAAV and the higher the concentration of non-rcAAV, the lower the probability that rcAAV will infect cells and replicate. Independent infection replicates of the same concentrations can help increase confidence that rcAAV will replicate in at least one infection replicate, but it is poorly understood how these concentrations change the probability of rcAAV replication. Therefore, we tested how the probability of detecting rcAAV changed with varying concentrations of wtAAV spiked into non-replicating rAAV.

Methods: We performed replication-based assays for rcAAV using wild-type AAV2 as a positive control and rAAV as a non-rcAAV. HEK-293 cells were infected with varying concentrations of wtAAV and rAAV along with a set concentration of Adenovirus Type 5 (Ad5) as a helper virus to allow for wtAAV replication. After an incubation period, the infected cells were harvested, lysed, and heat-inactivated to destroy Ad5. Lysates from the first infection are used to infect new cells for a second round of infection with a fresh addition of Ad5. This is repeated for a third infection round. After the third infection, qPCR was used on digested cells to detect the Rep2 gene. High concentrations of Rep2 after serial infections indicate replication of wtAAV.

Results: We first analyzed the probability of positive controls replicating without any interfering non-rcAAV particles. See the summary table for an overview of the results. At 1e7, 1e5 and 1e3 IU/mL, 100% of infection replicates showed replication in the assay. Lower concentrations of 1e2 and 1e1 IU/mL showed replication in 50% of infection replicates. We then used the 1e3 IU/mL concentration of wtAAV and added non-rcAAV particles in increasing concentrations. We found that as the concentration of interfering non-rcAAV particles increased, the probability of wtAAV replicating decreased. The probability of replication with 1e10, 1e11, and 1e12 vg/mL non-rcAAV was 100%, 85.7%, and 61.5%, respectively. These data show that as the concentration of rcAAV decreases, the probability of detecting viral replication decreases. Further, as the concentration of interfering non-rcAAV particles increases, the probability of rcAAV replication decreases.

Conclusion: These data underscore the importance of testing multiple infection replicates to determine whether rcAAV particles are present in a sample. Testing higher concentrations of sample is preferable due to the ability to screen more viral particles for rcAAV, but interference effects may mean that abundant non-rcAAV particles could block the ability for rcAAV to replicate and could lead to false negatives where rcAAV is present in the sample but is not able to infect cells and replicate. Method development and validation should be used to characterize the probability of this interference for a given combination of cell type, viral serotype, positive control concentration, and sample concentration. This information should be used to inform how many infection replicates are needed to be confident that rcAAV is not present when a result is negative. The probability of infection and replication will also depend on the interaction between a viral serotype and a host cell. For example, using AAV2 as a positive control is not the best choice for testing an AAV9 viral product as rcAAV of AAV9 will not have the same probability of infection as AAV2. We recommend that an ideal positive control material should match the serotype of the viral drug being tested.


An overview of the replication-based cell assay for detecting rcAAV. Three serial infections are performed. Following the final infection incubation, cells are digested and viral DNA is quantified using qPCR.


A summary of the results is displayed in this table. Each cell shows how many infection replicates show replication over the total number of replicates tested. The percentage shown is the percent chance of detection from the observed replicates (infection replicates that show replication over the total number of replicates tested). The left column shows the probability of replication of wtAAV without any interfering virus. Columns to the right show an increasing concentration of interfering virus at a single wtAAV concentration.