Adeno-associated virus (AAV) vectors play a pivotal role in gene therapy, delivering therapeutic genes with high precision and safety. However, a significant challenge in the biomanufacturing of these vectors is the potential formation of replication-competent AAVs (rcAAVs), which can pose safety risks in clinical applications. To address this challenge, biomanufacturers can employ rapid quantitative PCR (qPCR) assays.

The rapid qPCR assay described in this tech note is a cell-based technology developed by SK pharmteco to monitor the emergence of rcAAVs. The company has also developed technologies to mitigate the risk of rcAAV formation. Together, these technologies can help manufacturers of gene therapies sustain high-throughput operations, improve quality control, and enhance the safety and efficacy of their products.

The production of recombinant AAVs involves the co-transfection of three plasmids into HEK293 cells: the rep-cap plasmid, the adenovirus helper plasmid, and the AAV genome plasmid. The rep-cap plasmid carries the AAV replication (rep) and capsid (cap) genes essential for AAV genome replication and capsid formation. The adenovirus helper plasmid provides essential helper functions for AAV replication and packaging, whereas the AAV genome plasmid contains the therapeutic gene flanked by inverted terminal repeats (ITRs). The triple-transfection method is efficient and scalable, enabling the production of various AAV serotypes by altering the cap gene within the rep-cap plasmid.

However, during recombinant AAV production, nonhomologous recombination between the AAV vector and packaging DNA can lead to the formation of rcAAVs. These replication-competent vectors, if not managed properly, can replicate in the presence of a helper virus, potentially leading to unintended infection and replication within host cells. The presence of rcAAVs in clinical-grade AAV vector preparations is not just a concern, but a significant and pressing issue, as their behavior in the host post-administration, especially in the presence of natural helper viruses, is not fully understood.

Advanced detection methods

To address the challenge of rcAAVs, stringent quality control measures and advanced diagnostic techniques are required. Traditional methods for detecting rcAAVs, such as PCR-based assays, are effective but labor intensive and unsuitable for high-throughput commercial manufacturing. Recent advancements include the use of single-molecule, real-time sequencing and AAV genome population sequencing to detect and characterize rcAAVs. These technologies offer high sensitivity and specificity, and they are capable of identifying diverse recombination events leading to rcAAV formation.

At SK pharmteco, a cell-based qPCR assay has been developed to monitor the emergence of rcAAVs. This assay uses rep2-specific primers/probes, followed by confirmation with cap gene–specific primers/probes, to detect rcAAV events at a limit of detection of 10 infectious units. The qPCR assay is integrated into the manufacturing process, enabling timely detection and mitigation of rcAAV formation to ensure patient safety.

Figure 1. This schematic shows the steps involved in generating recombinant AAV by the triple-transfection method using plasmid DNA.

The application of the cell-based qPCR assay at SK pharmteco has proven robust and reliable. Transient transfection, involving multiple steps—such as cell expansion, plasmid DNA-transfection reagent complex formation, and bioreactor transfer—leads to the generation of viral particles (Figure 1).

Figure 2. Experimental design of a replication-competent AAV assay. (A) Amplification of recombinant AAV in the presence of helper virus. (B) Quantitative PCR endpoint assay.

The qPCR assay monitors for rcAAV emergence through continuous rounds of propagation of harvest lysates in the presence of adenovirus (Figure 2). This method amplifies AAV genomes containing ITR–rep-cap–ITR sequences, increasing progeny virus through subsequent rounds of infection. Extensive testing validated the assay’s specificity and sensitivity. Specificity acceptance criteria ensured accurate identification of target sequences, with positive amplification detected only in positive controls and test particles (Table 1).

The assay’s ability to detect the lowest concentration of rAAV2 confirmed the detection limit, ensuring high sensitivity. The linearity of the assay was evaluated through quantitative PCR standard curves, verifying accuracy across a range of input concentrations.

A bridging study was also conducted to evaluate the assay’s applicability to other AAV serotypes. The study confirmed that the assay design suits various AAV serotypes, demonstrating its broad utility in viral gene therapy applications. Primers and probes for AAV2 were validated, ensuring reliable detection and quantification of rcAAV2. Compatibility with different adenovirus preparations further enhanced the assay’s flexibility and practicality.

Importance of results for gene therapy production

The ability to reliably detect and mitigate rcAAV formation is crucial for the safety and efficacy of gene therapy products. The presence of rcAAVs poses significant risks, including potential unintended replication and infection within the host. Advanced detection technologies, such as the cell-based qPCR assay, provide a robust solution to monitor and control rcAAV emergence during manufacturing. Ensuring the absence of rcAAVs in clinical-grade AAV vector preparations enhances the safety profile of gene therapy vectors, reducing the risk of adverse events in patients.

The scalability and high-throughput capability of the qPCR assay make it suitable for commercial manufacturing settings. This ensures that quality control measures can be consistently applied across large production volumes, maintaining the integrity and safety of gene therapy products.

In addition to advanced detection methods, SK pharmteco has implemented several strategies to mitigate the risk of rcAAV formation. Promoter rearrangement is one strategy, where the P5 promoter is positioned 3′ of the cap gene in the vector design. This selective promoter activity prevents unintended gene expression that could contribute to rcAAV formation. Incorporation of introns to exceed AAV’s packaging limit is another strategy, ensuring that intact contigs cannot be packaged, thus reducing rcAAV emergence.

Observational data revealed that configuring test article constructs with the P5 promoter positioned upstream of essential replication and capsid genes led to a higher risk of rcAAV formation. Conversely, positioning the P5 promoter 3′ of these genes significantly reduced this risk, supporting the influence of promoter placement on vector safety. These mitigation strategies, combined with advanced detection methods, provide a comprehensive approach to ensuring the safety and efficacy of rAAV-based therapies.

Future directions

The ongoing development and refinement of the assay design focuses on enhancing sensitivity and accuracy. Integrating adenovirus 5 (Ad5) earlier in the protocol and optimizing freeze/thaw cycles are potential adjustments to improve assay performance. Shifting from qPCR to droplet digital PCR for endpoint analysis could offer superior precision and sensitivity, which would be particularly useful in detecting low levels of target DNA.

Future studies will involve pilot testing these adjustments, systematic analysis of freeze/thaw cycle effects, and controlled experiments to confirm the benefits of early Ad5 addition. Further research will explore alternative promoter and gene configurations to robustly prevent rcAAV formation without compromising vector efficacy. These efforts aim to enhance the capability to safely and effectively utilize rAAV vectors in clinical settings.

At SK pharmteco, Oxana M. Tsygankova, PhD, serves as senior scientist; Lana Sweet, director of virology, cell and gene therapy; Dana Cipriano, global head of testing and analytics; and Brian Tomkowicz, PhD, head of viral vector R&D.

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