ddPCR to innovate replication-competent virus testing

The latest Droplet Digital™ PCR (ddPCR™) technology and kits could provide a time-saving solution to transform replication-competent virus (RCV) testing in gene therapy workflows, helping to ensure the safety of new therapeutics.

Gene therapy describes the treatment or prevention of disease by tackling the underlying genetic issue. This is accomplished via gene silencing, replacement, or modification, and demonstrates particular promise for treating rare genetic conditions such as hemophilia and spinal muscular atrophy (SMA)¹. In the case of SMA – which represents the leading cause of inherited infant death – promising data in younger pediatric patients highlight the radical potential of gene therapy approaches to protect patients with a historical lack of treatment options². In other indications, cell therapy approaches (replacing diseased or damaged cells with new ones) also take centerstage.

For Dipika Gurnani, a Product Manager at Bio-Rad Laboratories, the promise of these approaches is an everyday focus. “My passion has always centered around rare diseases that have lacked effective diagnosis and treatment options,” she explains. “My primary objective at Bio-Rad is ensuring that the products in our gene therapy portfolio reach end-users and help researchers to bring safe and efficacious therapies to the market.”

A replication-competent enemy

Copies of therapeutic genes are usually delivered to target cells via viral vectors that are engineered to prevent replication. The most used vector is the adeno-associated virus (AAV), though lentiviruses and retroviruses may also be used.

When developing any viral vector, ensuring the absence of RCVs is a key concern that must be addressed to meet regulatory requirements and ensure patient safety. “As the name implies, an RCV is capable of reproducing and generating viral particles in the host cell,” Gurnani says. “Their presence in gene therapies introduces a risk of uncontrolled replication, leading to adverse events and severe illness in patients; in extreme situations, RCVs can lead to irreversible consequences.” During manufacturing, RCVs are considered a contamination that can impact product quality and consistency. RCV testing is thus required at different stages throughout gene therapy development and manufacturing. The specific point of testing varies with regulatory guidelines and specific product attributes, but typical stages involving RCV testing include cell line development, vector construction, in-process testing, and final product release. Then, after all this, post-therapy surveillance is also required.

“In-process RCV testing typically uses cell culture-based approaches because these are called out in the regulatory guidance,” Gurnani explains. “The issue is that these approaches take up to 45 days to provide results and carry a risk of lab contamination.” The same guidance also allows for nucleic acid-based testing (NAT) approaches like quantitative polymerase chain reaction (qPCR) in cases where they are shown to give equivalent results to cell culture methods specifically at lot release. When using these methods, however, researchers must ensure that the sensitivity of the primers and probes is good enough to avoid cross-reactivity and false positives. “Validating these assays can be resource- and time-intensive, and NAT can also struggle to differentiate between encapsulated and free-floating plasmids,” Gurnani says. “This is particularly problematic when using AAV vectors, but the key advantage is that a negative result can save companies from investing large amounts of time into testing via cell culture approaches.”

The promise of ddPCR in replication-competent virus detection

Droplet Digital PCR (ddPCR) offers the opportunity to run many thousands of measurements per sample. This is a vast improvement on qPCR, which is the historic method of choice when it comes to NAT-based methods but provides only one measurement per sample. Moreover, qPCR methods rely on relative measurements, meaning that an established reference point must be achieved (usually by producing a standard curve). “Producing standard curves may not be considered challenging, but they can be tricky and expensive to maintain over long periods of time,” Gurnani explains. “This is especially true in the world of gene therapies, where time delays can increase the time that therapies take to obtain regulatory approvals and reach patients.”

With these factors in mind, it may come as no surprise that many gene therapy developers are turning to ddPCR to drive the rapid detection of RCVs in their products. In Gurnani’s words, “ddPCR is very sensitive and highly specific. We partition a sample into 20,000 droplets, and classify each droplet as positive or negative. If there is a molecule in the droplet, it lights up as positive and we can use Poisson statistics to correct for the co-occurrence of two pieces of DNA in the same droplet. The result is an absolute count of the number of molecules in the sample without needing a standard curve.”

Bio-Rad is a pioneer of ddPCR kits for safety and contamination testing, including tests for residual DNA quantification for HEK293, CHO and E.coli cell lines, and mycoplasma. The latest addition to their arsenal: the Vericheck ddPCR Replication-Competent Virus Kits, are capable of identifying the absence of replication-competent AAV or lentivirus in just eight hours. “The new kits offer a rapid and validated quantification method that should accelerate safety testing for gene therapy companies while sparing them valuable resources,” Gurnani explains.

As a provider of instruments and solutions Gurnani and her colleagues often encounter custom-made kits that have been developed by individual customers. Each customer tailors a specific set of primers and probes, rigorously testing them to ensure alignment with their Standard Operating Procedures (SOPs).

“Our goal is to offer standardized kits that can be universally accepted and compared across diverse laboratories and companies. This approach fosters regulatory acceptance, allowing regulatory agencies to confidently recognize and trust the consistency and reliability of these standardized kits,” Gurnani says. “In addition, we recently launched the six-color QX600™ Droplet Digital™ PCR System that is being used for vector integrity, and promises even more precise and reproducible results.” By making these kits and solutions accessible on a common platform, Bio-Rad is able to deliver additional value to its customers.

A whole new world of therapeutic testing?

Recent advances like the first CRISPR-based therapy approval in 2023 highlight the capacity for major advances to earn their place in contemporary science. Could ddPCR kits do the same? Bio-Rad believes so, but universality is one major hurdle to overcome. “Our goal is to offer standardized kits that can be used universally, and their results compared across labs,” Gurnani says. “We are dedicated to making these kits accessible on a common platform to deliver additional value for our customers. A standardized kit would also allow regulatory agencies to trust the consistency and reliability of the results obtained.”

As our testing approaches evolve, so do cell and gene therapies. Today, these approaches are moving beyond the realm of rare diseases and into the battle against other conditions, such as cancer. In fact, recent reports suggest that there are an estimated 1,300 active cell therapy trials targeting different cancers³. This transition is a gentle reminder of the overall goal of these therapies: to save patients. This goal is more apparent for many rare diseases, which still lack curative treatment. Advances like the ddPCR testing kits from Bio-Rad may catapult us towards solutions that can revolutionize and save the lives of patients around the globe.

References

Sayed N., et al., Gene therapy: comprehensive overview and therapeutic applications. Life Sci (2022).

Ogbonmide T., et al., Gene Therapy for Spinal Muscular Atrophy (SMA): A Review of Current Challenges and Safety Considerations for Onasemnogene Abeparvovec (Zolgensma). Cureus (2023).

Gustafson M.P., et al., Emerging Frontiers in Immuno-and-Gene Therapy for Cancer. Cytotherapy (2023).