Quantifying AAV Viral Titer and Integrity with ddPCR

Mark White - Associate Director of Biopharma Product - Marketing Digital Biology Group - Bio-Rad

Marwan Alsarraj - Biopharma Segment Manager - Digital Biology Group - Bio-Rad

As the COVID-19 pandemic and subsequent vaccine development showed us, viruses and their components can both cause disease and help prevent it. While genetic illnesses are not born from a viral infection, scientists have found in recent years that viruses like adeno-associated virus (AAV) and lentivirus might be the key to solving these stubborn diseases. Specifically, they serve as the primary delivery vehicle for treatment. After gutting them of their native genetic material, scientists exploit their infectious nature to plant therapeutic genetic sequences in patients’ cells to either augment or silence problem genes and restore normal function.

The two gene therapies in the clinic today, as well as the majority of gene therapies currently in development, use AAV to deliver healthy genetic material to patients.1 Gene therapy developers have embraced AAV for several reasons:

  • They are not pathogenic in humans
  • They promote the long-term expression of transgenes in somatic cells
  • They elicit minimal levels of inflammation
  • In some cases, they can direct immune tolerance to transgene products2

AAV-based gene therapy development is not straightforward, however. It requires meticulous skill at each step. First, the desired genetic payload sequence must be inserted into the virus, and the vector must transfect host cells and replicate. Then, the host cells must be lysed to allow the vectors to be harvested and then purified to remove host cell contaminants and empty AAV capsids.

It is challenging to remove cells and cell debris from a batch of AAV vectors without removing active viral particles. AAV particles tend to bind to cell debris, leading to low yields and high impurity levels.3 Furthermore, different AAV serotypes require different approaches to purification to yield high AAV levels while maintaining the vectors’ integrity. 

Low yields create a dosing problem. If the vector concentration in a gene therapy batch is too low, developers would have to increase the dose volume to an unreasonable level.3 For

example, therapies targeting neurological disease must be produced in small volumes to fit in the small compartments of the brain and spinal cord. These therapies must be delivered at a concentration of 1014 vectors per kilogram, which is about 500 times the concentration that is typically achievable following upstream bioprocessing of the vector.3 According to one study, developers must concentrate their AAV batches 100-10,000 times to reach an appropriate titer.4

Overall, these challenges create uncertainty in the AAV development process and could potentially hamper the efficacy and safety of gene therapies. For a gene therapy to succeed in clinical trials and make a difference for patients, developers need to quantify yield after both upstream and downstream bioprocessing and screen out batches that do not contain a high enough viral titer. For this testing, manufacturers need to use accurate and precise methods.

qPCR is Not Precise Enough to Measure AAV Titer

Relatively inexpensive and reliable for most research applications, quantitative PCR (qPCR) has historically been most developers’ technique of choice for AAV titer quantification. However, qPCR does not provide a sufficiently accurate measure of viral titer to ensure the safety and efficacy of gene therapies. Notably, the technique often produces an overestimation of the viral titer.

qPCR struggles to offer an accurate result because it relies on a standard curve for reliable PCR amplification. Developers begin with a sample containing an unknown concentration of vectors and count how many amplification cycles it takes to reach a certain threshold. This number, called the Cq, is translated into vector concentration using the standard curve. Manufacturers must prepare these standard curves manually, a process that is prone to human error that can lead to variability in the result.

Cq is an unreliable proxy for viral titer because of its dependence on a standard curve. A standard curve is typically produced using DNA reference plasmids, which can form secondary structures that prevent primers from binding. If the primers fail to bind to the reference standard, it will not amplify as much as expected. Due to impaired amplification of the reference standard, qPCR will yield a lower Cq Since a lower Cq correlates with a higher viral titer, this can cause a developer to overestimate viral titer in the original sample.2 In one study, researchers found that standard curve calibration can also vary depending on where on the reference gene the primer binds.

Since gene therapies are meant to treat disease in humans, there is no room for error. Therefore, gene therapy developers must adopt more reliable techniques for measuring viral titer. The right tool would not need calibration, but rather, it would quantify AAV titer directly. That tool might be Droplet Digital PCR (ddPCR).

Measuring AAV Titer with Droplet Digital PCR

Unlike qPCR, ddPCR quantifies nucleic acid sequences directly and therefore does not rely on a standard curve. The technique involves partitioning 20 μL of reaction mixture into approximately 20,000 uniform 1-nL droplets and performing a separate PCR reaction in each one. 

Each partition contains no more than a few nucleic acid strands, and when AAV manufacturers use a vector genome-specific primer, amplification will only take place in droplets that contain the vector genome sequence. As the DNA amplifies, sequence-specific probes are cleaved and release a signal that causes the vector genome-containing droplets to fluoresce. Then, the PCR cartridge is transferred to a digital reader that counts the number of fluorescent and non-fluorescent droplets. Finally, the software automatically calculates the AAV titer in the original sample using Poisson statistics.

According to research performed by Dr. Birei Futura-Hanawa and colleagues at the National Institute of Health Sciences in Japan, ddPCR is less sensitive to secondary structures in the target DNA. In one study of AAV titer, she and her team found that while secondary structures impeded amplification and affected her qPCR results, her ddPCR results were not sensitive to the presence of secondary structures. 

In one study, ddPCR technology quantified single-stranded AAV vector genomes with four times greater sensitivity than qPCR.6 In another study, Dr. David Dobnik, Associate Professor at the Institute of Biology in Slovenia, found that ddPCR technology quantifies AAV titer more precisely and robustly than qPCR.7 Across several samples from multiple steps of the purification process, ddPCR quantified AAV titer with a significantly lower coefficient of variation. In the same study, Dobnik found that ddPCR technology picked up the presence of free-floating DNA in his samples. After he treated his samples with DNAse, ddPCR picked up the degradation of these DNA strands, suggesting another use for the technology: testing the downstream bioprocessing workflow and finding ways to improve it.

Aside from the partitioning, ddPCR and qPCR employ the same PCR chemistry. The key difference between the two techniques – and what makes ddPCR more accurate and robust than qPCR – lies in how nucleic acids are measured. Since Cq is a scalar measurement, qPCR depends on consistent and reliable amplification of DNA standard to yield an accurate measure of viral titer. ddPCR, on the other hand, doesn’t depend as much on the efficiency of the PCR reaction. It provides a binary measurement of whether or not amplification took place in each droplet, regardless of the magnitude, and only then combines these data to yield an overall measure of concentration.

Viral Genomes Versus Infectious Genomes

Viral titer measurements generated using ddPCR technology provide a concise and accurate indication of viral titer and give AAV developers a reasonable idea of a gene therapy’s potency. However, the potency of a gene therapy depends not just on AAV titer but also on the vector genomes’ integrity. In fact, the concentration of viral genomes may differ from the concentration of infectious genomes;8 therefore, gene therapy developers cannot fully determine the potency of a therapy without assessing both metrics.

A gene therapy batch may contain degradation products, contaminant DNA, or truncated vector genomes, and it can be challenging to differentiate between these entities and full, functional vector genomes. A single PCR primer, for example, may bind off- target sequences, leading to an overestimation of infectious genome concentration. Ultimately, this could lead a developer to produce a therapy that does not contain enough infectious genomes to make it effective.

To overcome this critical limitation, Futura-Hanawa developed a two- dimensional (2D) ddPCR assay that incorporates two probes that make it easier to assess the integrity of vector genomes.

Measuring Vector Integrity and Activity

Futura-Hanawa’s 2D ddPCR assay measures whether vector genomes are complete by using two probes that bind to two distant regions of the AAV vector genome. If droplets emit signals from both probes, this indicates that the vector genome is present and complete. In her aforementioned study, Futura-Hanawa used this technique to discover that only roughly 60 percent of her AAV contained full genomes.2 

Additionally, since 2D ddPCR technology can detect incomplete genomes, Futura-Hanawa tested whether her technique could be used to predict the activity of AAV vectors by measuring the degradation of vector genomes. To model degradation in the body, Futura-Hanawa incubated AAV vectors at body temperature for several days. Using ddPCR to measure degradation and fluorescence-activated cell sorting (FACS) to measure activity, she found that AAV degradation correlated with reduced activity. She ran the same experiment using qPCR, but qPCR was not able to detect degradation as reliably: it could not predict AAV activity.

ddPCR technology could potentially incorporate more than two probes to offer a more complete picture of vector integrity. With greater accuracy in this metric, as well as in the measurement of viral titer, developers could more accurately determine the potency of the gene therapy batches.

The Future of Gene Therapy Development 

Compared to more traditional drug classes, gene therapies are significantly more complicated to develop. Developers must rely on the activity of viruses, the integrity of nucleic acids, and the sensitivity and specificity of numerous purification steps to create safe and effective therapies.

Unpacking the complexity of the process makes it clear that developers must monitor their workflows more closely. In contrast to qPCR, ddPCR technology’s ability to quantify AAV titer directly and detect the degradation of vector genomes makes it easier for developers to assess the potency of their batches. It is a reliable solution for developers of gene therapies since it provides absolute quantification of AAV titer and can predict potency before a treatment ever enters a human.

Gene therapy has been decades in the making, but with appropriate regulation, standardization, and quality control using tools like ddPCR technology, manufacturers can ensure that gene therapies perform as expected and find more success in clinical trials. Such reliability, in turn, will enable gene therapies to enter the clinic in greater numbers and bring much-needed relief to the millions of individuals for whom gene therapy might be their first and only hope. 

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. 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.
  3. 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/
  4. Hebben M. Downstream bioprocessing of AAV vectors: industrial challenges & regulatory requirements. Cell & Gene Ther Ins. 2018 Mar;4(2): 131-46.
  5. Wang F, Cui X, Wang M, Xiao W, Xu R. A reliable and feasible qPCR strategy for titrating AAV vectors. Med Sci Monit Basic Res. 2013 Jul;19: 187–193.
  6. Lock M, McGorray S, Auricchio A, Ayuso E, Beecham EJ, Bluoin-Tavel V, et al. Characterization of a recombinant adeno-associated virus type 2 reference standard material. Hum Gene Ther. 2010 Oct;21(10): 1273–1285.
  7. Dobnik D, Kogošek P, Jakomin T, Košir N, Tušek Žnidarič M, Leskovec M et al. Accurate Quantification and Characterization of Adeno-Associated Viral Vectors. Front Microbiol. 2019 Jul; 10: 1570.
  8. Ayuso E, Bluoin-Tavel V, Lock M, McGorray S, Leon X, Alvira MR, et al. Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material. Hum Gene Ther. 2014 Nov;25(10): 977–987.

Author Biographies 

Mark White is the Associate Director of Biopharma Product Marketing at Bio-Rad. He has played a key role in the development of multiple core technology capabilities and assays alongside a multidisciplinary team of biologists and engineers at Bio-Rad and previously at Berkeley Lights Inc. Mark obtained his Ph.D. in Biomedical Sciences at the University of California, San Francisco.

Marwan Alsarraj is the Biopharma Segment Manager at Bio-Rad. He has been at the forefront of developing, marketing and commercializing technologies in the past 15 years in the life science research industry. Marwan obtained his M.S. in Biology at the University of Texas, El Paso.

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