Gene therapy has surged in popularity over the past few years thanks to the development of relatively innocuous viral vectors such as adeno-associated virus (AAV) and lentivirus. AAV serves as the gene delivery vehicle for the two gene therapies currently on the market and the majority of those currently in development.¹ However, generating AAV-based gene therapies is a complex process. The upstream and downstream bioprocessing of AAV vectors has not been standardized or optimized, making it difficult to reliably generate sufficient yields to produce a potent therapy in a reasonable volume.² To ensure a gene therapy is potent enough, technicians must measure viral titer throughout the development process and screen out batches that do not contain a sufficient concentration of viral genomes.

Scientists use traditional DNA quantification tools such as quantitative PCR (qPCR) to answer a myriad of scientific questions, but given the imprecision of the AAV workflow, this technology is not sensitive or precise enough for use in the development of gene therapies. Rather, another form of PCR technology, called Droplet Digital PCR, offers the precision necessary to identify ineffective or unsafe batches early and ensure AAV-mediated gene therapies’ safety, effectiveness, and consistency.
 
The Challenge of Quantifying AAV Titer
On its own, AAV is ideally suited for gene therapy. This virus does not cause disease in humans, nor does it elicit significant levels of inflammation. Research suggests that it promotes the long-term expression of transgenes and sometimes directs the immune system to tolerate transgene products, which improves the chances a gene therapy will positively impact patients.³ Its infectious nature enables it to deliver healthy genetic information to human cells. However, manufacturing AAV-based gene therapies involves a complex protocol that is difficult to control.

A technician starts the process by inserting the desired genetic sequence into the virus to create functional vectors. Then, they use cells to amplify the virus and lyse the cells to harvest the vectors. Finally, the technician purifies the virus to remove contaminants. This is where the challenges begin: AAV particles tend to bind to cell debris, making it difficult to filter out the debris without filtering out vectors.² Technicians must vary their approach to vector purification depending on the AAV serotype they’re working with, further complicating the process. As a result, AAV-based gene therapies require significant technical skill to manufacture, and developers continually struggle to generate high yields.

If AAV titer is too low, developers must compensate by increasing the product volume; however, high volumes are not compatible with certain neurological indications, where concentrated therapies must be delivered to the small compartments of the brain and spinal cord.² Upstream bioprocessing typically does not yield viral titers high enough to satisfy this requirement. Physicians would need to deliver doses containing 10¹⁴ vectors per kilogram, which is about 500 times greater than what is currently achievable.² Consequently, developers need to concentrate their batches 100-10,000 times to reach an acceptable titer.⁴

Technicians must quantify viral titer after both the extraction and concentration steps to determine whether their batches contain a high enough viral titer. To do this effectively, manufacturers need access to an accurate and precise method for measuring the concentration of viral genomes in each batch.

qPCR is relatively inexpensive and reliable for most research applications, and it is a popular technique for AAV titer quantification. However, because of how it measures nucleic acid concentrations, this technique cannot quantify viral titer precisely enough to guarantee the potency of a gene therapy batch. qPCR enables a technician to estimate the viral genome concentration in a sample by counting the number of amplification cycles it takes to reach a pre-defined threshold. The more cycles it takes, the smaller the concentration in the original sample; therefore, the number of cycles can be translated into a vector genome concentration using a standard curve. However, two issues arise here that hamper the accuracy and precision of this result. First, manufacturers must generate this standard curve using serial dilutions, which is a time-consuming and unreliable process that often leads to variability in qPCR results. Second, the DNA standards used to create the standard curve might not amplify with the reliability needed to generate an accurate standard.

The DNA reference plasmids used to produce the standard curve don’t always replicate efficiently because secondary structures can form that prevent primers from binding to the DNA. If this occurs, the reference DNA will not amplify as much as expected, and by comparison, it will appear that the test sample contains a higher concentration of vector genomes.² One study found that qPCR results can vary based on the location on the DNA where strand the primer, as well.⁵ This variability cannot be tolerated in gene therapy development, where human lives are at stake. Therefore, developers must adopt more precise techniques to quantify viral titer.
 
Measuring AAV Titer with Droplet Digital PCR
ddPCR offers an alternative approach to AAV titer measurement that is more sensitive and precise. ddPCR does not rely on a standard curve; rather, it offers absolute quantification of nucleic acids. Before initiating PCR, a technician loads a cartridge containing 20 μL of reaction mixture into a droplet generator. This instrument uses oil-emulsion technology to partition the sample into approximately 20,000 1-nL droplets. Each droplet ultimately contains one or a few nucleic acids each.
           
Some droplets will contain the target nucleic acid sequence, and some won’t. In the droplets that do contain the target sequence, sequence-specific primers will cause these sequences to amplify, and a probe will cause these droplets to fluoresce. Meanwhile, if a droplet does not contain the target sequence, amplification will not take place. Instead, these droplets will only emit weak background fluorescence. Finally, a droplet reader counts the number of fluorescent versus non-fluorescent droplets and uses Poisson statistics to translate this ratio into a genome concentration.

ddPCR and qPCR use the same primers and probes, yet qPCR and ddPCR fundamentally differ in their approach to measuring nucleic acid concentration. qPCR is reliable for detecting nucleic acids because detection only requires that amplification take place to some degree; if DNA amplifies, a technician knows their target nucleic acid is present. However, when a technician must measure DNA concentration, such as in AAV titer quantification, qPCR technology becomes less reliable because it requires more precisely controlled amplification. ddPCR technology, on the other hand, does not depend on consistent amplification. Rather, it takes a digital measurement of vector genome-positive droplets to calculate the nucleic acid concentration in the original sample.This approach makes ddPCR more accurate than qPCR.

ddPCR technology outperforms qPCR on multiple parameters. First, researchers have found that, compared to qPCR, ddPCR is less susceptible to interference from secondary structures in the target DNA. In one study, secondary structures impaired a qPCR-based measurement of AAV titer, but not the measurement produced by ddPCR technology.³ In another study, researchers found that ddPCR technology was four times more sensitive than qPCR in the quantification of single-stranded AAV vector genomes.⁶ ddPCRis also more precise and robust than qPCR.⁷ One research team analyzed several samples taken from various steps of the purification process and found thatddPCR quantified AAV titer with a significantly lower coefficient of variation.⁷ In the same study, ddPCR technology detected the degradation of free-floating DNA using DNase. This indicated the importance of DNase treatment and demonstrated the role of ddPCR technology in refining purification protocols.
 
Going Beyond Titer: Measuring AAV Integrity
ddPCR technology precisely and accurately measures viral titer, providing developers with an approximation of a gene therapy’s potency. However, viral titer does not necessarily equal the concentration of infectious vectors;⁸ therefore, developers must also measure the functionality of their vectors to gather a complete potency measurement.

Some assays may not differentiate between full, functional vectors and contaminants such as degradation products, contaminant DNA, or truncated vector genomes. As a result, a primer could bind to contaminating sequences and cause technicians to overestimate potency, leading them to produce a therapy that is not effective. To measure AAV titer and vector integrity simultaneously, Birei Futura-Hanawa, Ph.D. developed a duplex ddPCR assay, termed 2D ddPCR, that makes it possible to quantify full vector genomes.

Futura-Hanawa’s 2D ddPCR assay uses two probes to determine whether an AAV genome is complete. The two probes bind to distant regions of the genome, and if droplets emit signals from both, this indicates the genome is likely complete and the vector is likely active. Testing her AAV vectors using this method, Futura-Hanawa found that roughly 40% of her AAV vectors contained incomplete genomes.³ This suggests that by only using one probe, one could vastly overestimate the concentration of active vectors in a sample.

Next, Futura-Hanawa hypothesized that her 2D ddPCR assay could detect AAV genome degradation in the body and predict changes in AAV activity over time. To test this, Futura-Hanawa modeled degradation in the body by incubating AAV vectors at body temperature for several days. Her team used ddPCR and qPCR to measure changes in genome integrity and used fluorescence-activated cell sorting (FACS) to measure AAV activity. They found that these two measures were correlated only when genomes were counted using ddPCR. This study indicated that liquid biopsies based on 2D ddPCR technology could potentially be used to monitor AAV activity in the body.

An assay like this doesn’t have to stop at two probes, however. Incorporating more probes into this test could provide researchers with even greater specificity. This, in turn, would provide developers with a more accurate picture of their therapies’ potency.
 
More Streamlined Gene Therapy Development
For gene therapy production to be sustainable, developers must reduce waste by ensuring their batches are safe and effective before and during clinical trials. ddPCR is more sensitive and accurate than qPCR, making it an ideal tool for monitoring AAV vector potency and aiding developers in screening out failed batches. With more streamlined quality control, gene therapy developers can bring millions of patients new hope for longer and higher-quality life.
 
References
1.     Clinicaltrials.gov. Search: “gene therapy+aav” — Recruiting, Active, Not Recruiting Studies — List Results [Internet]. Washington, DC: US National Library of Medicine; 2021 [updated 2021 May 18; cited 2021 May 18]. Available from: https://clinicaltrials.gov/ct2/results?term=%22gene+therapy%22+%22aav%22&Search=Apply&recrs=b&recrs=a&recrs=d&age_v=&gndr=&type=&rslt=
2.     Hernandez Bort J. Challenges in the Downstream Process of Gene Therapy Products. American Pharmaceutical Review [Internet]. 2021 Feb 19 [cited 2021 May 18];Article Archives:[about 5 p.]. Available from: https://www.americanpharmaceuticalreview.com/Featured-Articles/362178-Challenges-in-the-Downstream-Process-of-Gene-Therapy-Products/
3.     Furuta-Hanawa B, Yamaguchi T, Uchida E. Two-dimensional droplet digital PCR as a tool for titration and integrity evaluation of recombinant adeno-associated viral vectors. Hum Gene Ther Methods. 2019 Aug;30(4): 127-136.