Inadequate quality control assays shouldn’t limit cell and gene therapy applications. Instead, quantifying the potency of cell and gene therapies is essential for successful therapeutics. In this article, we describe multiple leading potency assays developed using Droplet Digital PCR (ddPCR) technology, including an assay that quantifies transgene integration and expression in one step.
Cell and gene therapies are ushering in a world where treatments are specific to an individual’s genetics. Cell therapies such as chimeric antigen receptor (CAR) T cells take patient-derived or pooled cells and modify them outside the patient. Then the modified cells are reintroduced as treatment. Gene therapies alter the DNA inside target cells while still within a patient to alter the expression of pathological genes. Gene therapy can enhance expression through augmentation or eliminate it through gene silencing.
The final gene dosage a patient receives from cell or gene therapy is critical to the success of the treatment and the patient’s safety. Designers of cell and gene therapies walk a razor’s edge between life-saving treatment and making matters worse. Therefore, testing the potency of cell and gene therapies is critical. Here we explore case studies where ddPCR technology enables effective and efficient potency testing in cell and gene therapy applications.
What Is Potency Testing?
The U.S. Food and Drug Administration (FDA) defines potency as “the specific ability or capacity of the product, as indicated by appropriate laboratory tests or adequately controlled clinical data obtained through the administration of the product in the manner intended to effect a given result” (U.S. Department of Health and Human Services 2011). The complexity of cell and gene therapy can make quantifying potency tricky.
For cell therapies, heterogeneity in donor material or within cell lines can cause batch-to-batch variability. Meanwhile, gene therapies have two steps that require potency quantification: 1) transference of genetic material and 2) target gene expression. The FDA recommends that “potency assays … represent the product’s mechanism of action (i.e., relevant therapeutic activity or intended biological effect).” These one-two-punch assays can be technically challenging to design.
However, as the case studies below demonstrate, ddPCR assays offer the absolute quantification and consistency required to accurately measure cell and gene therapy potency.
AAV Testing with ddPCR Technology
The adeno-associated virus (AAV) is gene therapy’s most common DNA delivery system. The clinical dosage is presented as vector genome titer per milliliter and is typically quantified by quantitative PCR (qPCR). However, qPCR requires a standard curve, is sensitive to inhibitors, and is prone to batch-to-batch variability.
Two research groups (Dobnik et al. 2019 and Furuta-Hanawa et al. 2019) used ddPCR technology to quantify AAV vector genome copies while circumventing these issues. The authors reported that ddPCR assays outperformed qPCR in specificity, accuracy, and consistency. While the authors correlated AAV titer with biological activity, they did not quantify potency in a biological system. The next case study overcomes this limitation with a novel ddPCR method.
Quantifying Integration and Action in One Step
As described above, the ideal gene therapy potency test would simultaneously quantify the transfer of genetic material and changes in expression. In their 2021 publication, Clarner and colleagues did just that (Clarner et al. 2021). The authors simultaneously measured vector integration and effect using a one-step reverse transcription Droplet Digital PCR (RT-ddPCR) method. Additionally, they validated their assay in vitro and in vivo using non-human primate models.
The authors found that their one-step RT-ddPCR method quantified transgene expression and potency with RNA interference (RNAi) and augmentation vectors. The results showed impressive consistency and correlated well with validating enzyme-linked immunosorbent assays (ELISAs). The researchers noted that the ddPCR workflow provides absolute quantification of target genes without standard curves, reducing variability. Beyond one-step quantification, ddPCR technology provided a more straightforward workflow with greater robustness, sensitivity, and precision than alternative methods.
Don’t Forget Cell Therapy
While ddPCR methods are excellent for gene therapy potency testing, they are also ideal for cell therapy transgene quantification. To generate CAR T cells, scientists use viral vectors to insert transgenes into the DNA of T cells. However, the number of transgene copies integrated into a cell’s DNA can vary. A higher number of transgenes suggests greater potency but increases the risk of genotoxicity.
Lu et al. 2020 used ddPCR technology to measure transgene copy number in genetically engineered CAR-T cells. They found that the ddPCR workflow was robust for copy number quantification with high reproducibility. In fact, ddPCR assays accurately measured transgene copy numbers in frozen samples and with different technicians.
Droplet Digital PCR Puts Precision in Precision Medicine
Cell and gene therapies are poised to revolutionize the clinic. However, they’re only as practical as their quality control allows. When gene dosage is the critical therapeutic parameter, quantifying potency is essential.
Across studies, ddPCR technology has emerged as the optimal potency quantification technique because it combines the optimal qualities of absolute quantification, unparalleled consistency, and high accuracy. As a result, ddPCR workflows are paving the way to precision medicine with precisely quantified potency.
Clarner, P et al. (2021). Development of a one-step RT-ddPCR method to determine the expression and potency of AAV vectors. Mol Ther Methods Clin Dev 23, 68–77.
Dobnik D et al. (2019). Accurate quantification and characterization of adeno-associated viral vectors. Front Microbiol 10, 1570.
Furuta-Hanawa B et al. (2019). Two-dimensional droplet digital PCR as a tool for titration and integrity evaluation of recombinant adeno-associated viral vectors. Hum Gene Ther Methods 4, 127–136.
Lu A et al. (2020). Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell products. J Transl Med 1, 191.
U.S. Department of Health and Human Services, U.S. FDA (2011). Guidance for industry: Potency tests for cellular and gene therapy products. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/potency-tests-cellular-and-gene-therapy-products, accessed June 23, 2022.