Gene therapy is primed to become the next major development in medicine and bring relief to patients living with diseases ranging from haemophilia to Alzheimer’s disease, write Mark White, Associate Director of Biopharma Product Marketing, and Marwan Alsarraj, Biopharma Segment Manager, Bio-Rad
More than four hundred gene therapy trials are recruiting or active in the US right now.1 However, as evidenced by decades of research, gene therapy development is not straightforward.
For example, a significant challenge to developing gene therapies is producing doses that contain the correct concentration of healthy gene copies, which dictates the potency of the dose.
The primary delivery vehicle for healthy gene copies is the adeno-associated virus (AAV); but, AAV titre is not easy to measure accurately, which creates uncertainty in the vector genome concentration and, in turn, the safety and efficacy of each dose.
AAVs are the most common vectors used in gene therapies because they are naturally safe and effective carriers of genetic information. They do not cause illness in humans; they persistently express transgenes in non-dividing cells; they possess a low immune profile, in that they only elicit a limited immune response; and, in some cases, they also direct the immune system to tolerate transgene products.2
Despite these advantages, AAV development presents challenges for manufacturers. One of the most prevalent difficulties is producing functional vectors in high concentrations.
After the upstream bioprocessing of AAV vectors, the vector concentration in crude harvests could reach 2 x 1011 vector genomes/mL, but this concentration is not sufficient to create a dose in a reasonable volume for use in patients.3
Liver-directed gene therapies such as those for haemophilia B, for example, must be administered at a dose of 1012 vector genomes/kg. Meanwhile, therapies targeting smaller compartments, such as the central nervous system (for conditions like Alzheimer’s disease), must be administered at a higher concentration of 1014 vectors/kg to reduce the dose volume.3
To deliver effective doses for these and other conditions at a reasonable volume, developers must concentrate their AAV vectors 100 to 10,000 times.4 This discrepancy requires that developers accurately quantify AAV titre after both the purification and concentration steps to ensure that their final product contains the correct vector dose. To do so, manufacturers must adopt reliable testing methods.
The most common method used to measure AAV titre quantifies vector genomes using quantitative PCR (qPCR).
Although suitable for many applications, qPCR is not precise enough to assure manufacturers of the potency of their AAV batches because it relies on a relative measure: to determine AAV titre using qPCR, manufacturers count the number of amplification cycles it takes to reach a threshold determined using a standard curve.
Manufacturers prepare these standard curves using a reference material, typically plasmid DNAs, but these reference standards are not always reliable.
For example, the reference plasmid DNA might form secondary structures that affect qPCR results. The primer can struggle to bind to the reference standard, which hampers amplification and produces an overestimation of viral titre.2
Also, one study found that qPCR calculated different titres depending on where on the reference gene the primer bound.5
To overcome these limitations, Dr Birei Futura-Hanawa and her team at the National Institute of Health Sciences in Japan developed a two-dimensional (2D) droplet digital PCR (ddPCR) assay that quantifies AAV titre without a standard curve.
ddPCR uses partitioning technology to quantify nucleic acid sequences. The method begins by loading 20 μL of reaction mixture into cartridges. It then gets divided into approximately 20,000 uniform 1 nL droplets that each contain no more than a few nucleic acid strands.
A separate PCR reaction takes place in each one. As the DNA amplifies, sequence-specific probes are cleaved and release a fluorescent signal that lights up the droplet.
AAV manufacturers use primers that are specific to the AAV vector; therefore, as the reaction takes place, only the vector genome will amplify. This means droplets that contain the AAV genome will emit a strong fluorescent signal, whereas droplets that do not contain the AAV genome will only emit weak fluorescence.
After the PCR step, a digital reader counts the number of fluorescent and non-fluorescent droplets. Using Poisson statistics, the software automatically calculates the AAV titre in the original sample.
As ddPCR does not rely on a standard curve, it is not prone to calibration errors. Further, Dr Futura-Hanawa found that ddPCR is less sensitive to secondary structures in the target DNA than qPCR.2
Another factor that can affect the potency of a treatment is the proportion of fully functional vectors in the batch. Viral genome concentration and infectious genome concentration can differ, sometimes because of inaccuracies in how viral genomes are counted.6,7
An assay, for example, might detect degradation products formed during the purification and extraction of the genome, contaminant DNA or truncated vector genomes.
If an assay detects these non-functional genome sequences, it could overestimate the concentration of active vectors and lead a manufacturer to produce a therapy that insufficiently potent.
Not only does ddPCR offer a more accurate measure of viral titre than qPCR, but unlike qPCR, it can also measure AAV integrity and predict its activity in the body.
Futura-Hanawa’s 2D ddPCR assay detects whether vector genomes are complete by using two probes that bind to two distant regions of the AAV vector genome. Using this technique, Futura-Hanawa found that her vector batch contained roughly 40% incomplete genomes.2
Additionally, Futura-Hanawa found that her team’s 2D ddPCR assay could be used to predict vector activity in the body by quantifying the degradation of the vector.
After incubating the vectors at body temperature, the team found that degradation, as measured by quantifying vector genomes using ddPCR, correlated with vector activity. By contrast, they did not find any relationship between degradation and activity when they quantified degradation with qPCR.
Future development may lead to the production of multiplex assays that examine more than two regions of the genome to further increase the accuracy of this method to detect functional vector genomes.
Gene therapies are not easy to develop; the biopharmaceutical industry still has more to learn about how these products form at the cellular level.
In the meantime, as these therapies are being administered to patients, AAV developers must calculate the potency of their therapies by measuring AAV titre accurately and precisely.
ddPCR offers a reliable solution to developers of gene therapies as it provides absolute quantification of AAV titre and can predict potency before a treatment ever enters a human.
Incorporating ddPCR into the quality control process can infuse gene therapy development with greater certainty and lead to the production of more safe and effective treatments.