In this interview, Mark White (Bio-Rad Laboratories, CA, USA) discusses the range of cell therapy applications that are using droplet digital (ddPCR) technology, the advantages and challenges they face in developing accurate assays for lentiviral quantification and alternative approaches for the process. In addition to this, we hear his thoughts on the future of lentiviral quantification and the new assays and kits currently being developed by Bio-Rad to support researchers working on cell therapies.
What are the main challenges in developing an accurate assay for lentiviral quantification?
There are two main challenges faced when trying to accurately quantify lentiviral particles or the vector copy number of transduced cells. The first is the inherent variability of the entire stack up of instrument, process and operator. This can lead to coefficients of variability in the 20-50% range when comparing runs, instruments or operators. The second challenge is the need to maintain reference material in order to run a standard curve for quantification. This material must be very exact and it is costly to make, maintain and transition from one lot to another.
What are the advantages of using ddPCR over alternative approaches, such as qPCR or NGS, for accurate viral vector quantification?
The first key advantage of ddPCR for accurate viral vector quantification is the fact that ddPCR is an absolute count of DNA molecules, eliminating the need to use and maintain reference material for a standard curve. This saves time and money as well as freeing up about 25% of the valuable real estate on a well plate. In addition, ddPCR is much more tolerant to common inhibitors that are present in the matrices used in the viral vector process.
What are the most common pitfalls to avoid when transition from qPCR or NSG to ddPCR?
One of the most common pitfalls is the assumption that qPCR and ddPCR should produce the exact same viral titer or vector copy number when testing the same sample. The two methods, while based on the same method of PCR, have many differences in process and experimental factors, as well as assumptions about how each method performs. For example, if the reference material for a qPCR standard curve is not quantified accurately, this can ripple through the rest of the experiment and affect the final viral quantification. In addition, while many qPCR primer sets can be transitioned to ddPCR, they may need some optimization for annealing/extension temperature as well as the addition of a restriction enzyme in order to increase amplicon accessibility.
Can you tell us more about the range of cell therapy applications that are using ddPCR technology?
ddPCR is currently being used in a wide range of cell therapy applications. One of the most common is to estimate the vector copy number per cell by measuring the number of reference gene molecules to estimate the number of diploid cells, then measuring a component of the viral vector to estimate vector copies. In addition, a recent ddPCR workflow addition enabled encapsulation of a whole, live cell in a ddPCR droplet, followed by lysis and ddPCR to quantify the DNA present in each cell. This allows a researcher to quantify the % of cells that contain a viral vector. Monitoring contaminating DNA from host cells like HEK293, as well as replication competent gene machinery has also been used. Finally, ddPCR can be used to monitor gene expression of the viral vector in target cells, as well as track the viral vector counts in patient samples..
Are there any solutions that are already available for targeting specific cell therapies?
Bio-Rad currently has two expert design assays that target CD-19 CAR-T constructs. One assay targets the axicabtagene ciloleucel (axi-cel, sold under the brand name Yescarta) construct and a second assay targets a sequence that is shared by both the axi-cel and tisagenlecleucel (tisa-cel) constructs.
In your opinion, where do you see the future of lentiviral quantification heading?
I see a big push to quantify multiple components of the lentiviral construct using a multiplexed assay. The therapeutic effect of a lentiviral based therapy relies on the gene of interest sequence as well as the promoter, enhancer and polyA tail sequences on either side. You may have the gene of interest, but if the rest of the construct is truncated in some way, the therapy will be less likely to work. I see therapeutics developers starting to measure the titer or copy number of multiple vector components, as well as interest from regulatory bodies in defining the therapeutics more thoroughly with these multiplexed approaches.
What is Bio-Rad developing in the near future for cell therapies?
Bio-Rad is constantly developing new assays and kits to support the researchers working on cell therapies. We have recently launched a new cell and gene therapy assay design engine that helps researchers design fully custom and ddPCR optimized assays in a matter of minutes to any gene sequence. Next year, we are excited to be releasing a new product in our Vericheck family of QC kits.
How can people contact you if they would like more information?
The opinions expressed in this interview are those of the interviewee and do not necessarily reflect the views of RegMedNet or Future Science Group.
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