Biotech
Monday, July 25th, 2022 2:00 am EDT
Cell and gene therapies are taking the world by storm. With CRISPR gene-editing technology, the genome is no longer an untouchable sacred text. Instead, genomic DNA is actively manipulated in the laboratory and the clinic. This revolution is changing the bench, the bedside, and the boardroom. As a result, the cell and gene therapy market is valued at USD 4.99 billion and is expected to grow to over USD 36 billion by 2027.
The potential of personalized medicine––where treatments are specific to an individual’s genetics––explains this exponential growth. Cell therapies, such as CAR-Ts, modify cells outside the patient and reintroduce them as a targeted treatment. In comparison, gene therapies alter the DNA within cells. The final genetic dosage from a cell or gene therapy walks a razor’s edge between therapeutic and toxic. This makes the quality control testing of cell and gene therapies absolutely vital. Many turn to qRT-PCR for this task, but this method cannot deliver absolute quantification as each run requires a standard curve, creating inconsistencies.
In this article, we explore how Droplet Digital™ PCR (ddPCR™) technology is revolutionizing quality control for cell and gene therapies, enabling scientists to save time, money, and resources through confident quantification.
What is ddPCR technology?
Before we dive into how ddPCR assays reimagine the limits of quality control in cell and gene therapies, let’s explore ddPCR technology. Like qRT-PCR, ddPCR technology requires a nucleic acid sample. However, in ddPCR assays, samples are partitioned into 20,000 discrete droplets. Individual PCR reactions occur within each droplet, which on average contains just a single copy of the donor DNA. During PCR cycling, positive droplets that contain the target DNA will produce a fluorescent signal. This digital system results in absolute quantification without the need for standard curves, leading to unparalleled precision, simplified workflows, and removal of PCR bias.
ddPCR assays make cell and gene therapies possible
Generating cell and gene therapies is complex, requiring precise quality control. CRISPR, while powerful, does not have a 100% success rate. However, you need 100% certainty in your product. That’s where the absolute quantification from ddPCR technology comes in. Here we explore several instances where ddPCR assays allow scientists to expand cell and gene therapy boundaries.
The ideal quality control assay for gene therapy would simultaneously quantify the transfer of genetic material and changes in expression. In their 2021 publication, Clarner et al. did just that using a one-step RT-ddPCR method. Their method quantified transgene expression and potency with both RNAi and augmentation vectors in vitro and in vivo, using non-human primate models. They noted that the absolute quantification from ddPCR reduced variability and provided a more streamlined workflow.
In a second example, CRISPR can generate knock-ins to increase gene dosage, which is technically challenging because integration efficiency is low. The process of developing knock-ins becomes time and resource-intensive and requires processing many failures. CRISPR SNIPER uses ddPCR technology to quantify knock-ins with precision and efficiency. Because ddPCR assays partition donor DNA into 20,000 droplets, CRISPR SNIPER can accurately measure low-frequency integration events that qRT-PCR simply cannot detect.
Finally, ddPCR methods are excellent for cell therapy transgene quantification. CAR-T cells are generated using viral vectors that insert transgenes into T-cell DNA. However, the number of integrated transgene copies can be variable. Lu et al. used ddPCR technology to accurately measure transgene copy insertion in CAR-T cells with high levels of reproducibility.
Across the board, ddPCR assays are uniquely positioned to catapult cell and gene therapies to the next level, standardizing and safeguarding the production process worldwide. ddPCR methods possess the accuracy, precision, and reproducibility required for cell and gene therapy quality control. Across applications from knock-in generation to CAR-T cell transgene quantification, this technology allows scientists to focus on what they do best: science. Meanwhile, ddPCR technology can take care of quality control.
Learn more about ddPCR applications for cell and gene therapy manufacturing.
This post has been syndicated from a third-party source. View the original article here.
