Making gene therapy affordable with droplet digital PCR
Gene therapy has the potential to bring relief to millions of individuals with diseases ranging from β-thalassemia to sickle cell disease across Asia and beyond. This novel treatment modality is showing tremendous economic potential. According to one analysis, the global gene therapy market is expected to reach $3.3 billion by 2027 and enjoy a post-COVID-19 compound annual growth rate of 19.5 per cent.
However, gene therapy poses several unique technical challenges that must be overcome before becoming widely available and affordable. For example, gene therapy depends on inserting a healthy genetic sequence into a patient using a vector such as adeno-associated virus (AAV), but it is difficult to produce a therapeutic batch that contains enough vectors for the therapy to benefit patients.
Upstream bioprocessing of AAV vectors often yields crude harvests containing too low vector genomes per milliliter, to create a potent therapy in a reasonable volume. Therapies that target smaller compartments in the body, such as the central nervous system, where the volume must be reduced, require a vector concentration that is much higher. This means developers must concentrate their AAV vectors 100-10,000 times.
This enrichment process infuses development with uncertainty, creating a need for rigorous monitoring. Specifically, developers need to gain a precise understanding of their treatment’s potency. This requires the accurate quantification of AVV titer, after both the purification and concentration of the vectors, and the vector genome’s integrity.
Quantitative PCR (qPCR) is the most common method for measuring AAV titer, yet the method is not sensitive enough to determine the potency of a gene therapy. qPCR measures AAV titer based on a standard curve, which is not always reliable and can cause an overestimation of viral titer. Meanwhile, qPCR cannot measure the integrity of vector genomes and it thereby provides an incomplete picture of a batch’s potency. The limitations of qPCR in viral titer and integrity measurement create a need to adopt more versatile and reliable quality control techniques during AAV-mediated gene therapy development.
Droplet digital PCR (ddPCR) is an alternative to qPCR that quantifies nucleic acids directly, eliminating the calibration errors that come with using a standard curve. Since it measures nucleic acids directly, it overcomes the limitations of qPCR and provides a more accurate count of viral titer and integrity.
ddPCR involves partitioning 20 microlitre of reaction mixture into approximately 20,000 uniform 1-nano litre droplets that contain no more than a few nucleic acid strands each. Then, a separate PCR reaction takes place in each droplet.
As the DNA amplifies, sequence-specific probes are cleaved and release a signal that makes the droplets fluoresce. When gene therapy developers use AAV-specific primers, only the vector genomes will amplify, which means only the AAV genome’s droplets will emit a strong fluorescent signal. The droplets that do not contain the target sequence only emit weak fluorescence. After the reaction ends, a digital reader counts the number of fluorescent and non-fluorescent droplets one by one, and software uses Poisson statistics to automatically calculate the AAV titer in the original sample.
For instance, Dr Birei Futura-Hanawa and her team at the National Institute of Health Sciences in Japan have recently developed a two-dimensional (2D) ddPCR assay that not only quantifies AAV titer but also predicts the integrity of the vector genomes, thereby providing a more complete potency calculation than qPCR assays.
Additionally, by combining 2D ddPCR and fluorescence-activated cell sorting (FACS) and incubating her vectors at body temperature, she was able to correlate vector degradation with reduced activity. She did not see the same correlation when she used qPCR to quantify vector genomes.
As gene therapies become more common, quality control will become even more valuable. By providing a more precise measure of AAV titer and integrity, ddPCR can help developers gain confidence in their products and help them develop therapies that will help more people.