qPCR as a Tried-and-True Potency Quantification Method
For decades, qPCR assays have been considered the gold standard for nucleic acid quantification. This has included the measurement of cell therapy potency since the invention of this therapeutic modality. qPCR-based gene expression assaysare especially useful for examining the in vitro activity of CAR T cells and other cell therapies (Stroncek et al. 2007). In addition, individual potency biomarkers can be multiplexed to offer a fuller picture of immune cell activity.
Similarly, qPCR assays are a proven, inexpensive option for quantifying gene therapy potency. In particular, 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 researchers have typically used qPCR assays to quantify genome titer and gain important validation information about each batch of these promising therapeutics.
Stepping Up Sensitivity with ddPCR Technology
While scientists have historically leveraged qPCR for potency testing and other molecular quantification experiments, many are choosing to develop new workflows that use ddPCR assays to obtain even higher sensitivity and absolute quantification. For example, the Project A-Gene Report declared ddPCR technology the gold standard for measurement of AAV vector titer (Alliance for Regenerative Medicine and National Institute for Innovation in Manufacturing Biopharmaceuticals, 2021).
For example, in 2019, two research groups used ddPCR technology to quantify AAV vector genome copies with high specificity, accuracy, and consistency (Dobnik et al. 2019 and Furuta-Hanawa et al. 2019).
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. 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 straight forward workflow that offered robustness, sensitivity, and precision.
ddPCR methods are also ideal for cell therapy transgene quantification. In one 2020 paper, researchers used ddPCR technology to measure transgene copy number in genetically engineered CAR T cells (Lu et al. 2020). They found the ddPCR workflow to be robust for copy number quantification with high reproducibility. In fact, ddPCR assays accurately measured transgene copy numbers in frozen samples and with different technicians.
Putting "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, sensitive qPCR and ddPCR technologies have emerged as the optimal potency quantification techniques because they combine the optimal qualities of reliability and high accuracy to deliver trusted results. As a result, qPCR and Droplet Digital PCR workflows are paving the way to precision medicine with precisely quantified therapeutic potency.
References
- Alliance for Regenerative Medicine and National Institute for Innovation in Manufacturing Biopharmaceuticals (2021). Project A-Gene: A case study-based approach to integrated QbD principles in Gene Therapy CMC programs. http://alliancerm.org/wp-content/uploads/2021/09/ALL-PROJECT-A-GENE-V10.pdf, accessed December 30, 2022.
- 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.
- Stroncek et al. (2007). Potency analysis of cellular therapies: the emerging role of molecular assays. J Transl Med 5, 24.
- 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 December 30, 2022.
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