The Lurking Danger

Gene therapies can potentially transform the lives of people living with a wide range of chronic and terminal conditions. Since the first gene therapy product gained FDA approval in 2017, more than 20 others have been approved (1), with 20–60 more expected to reach 350,000 patients within the next decade (2).

Pathologists play an essential role in gene therapy development by ensuring the safety and integrity of these potentially life-saving therapeutics. Today, most gene therapies leverage adeno-associated virus (AAV) as their vehicle for nucleic acid delivery – but, because AAVs are grown in cell culture, gene therapy batches are susceptible to contamination, particularly by Mycoplasma species.

Nearly one in three cell lines across the world are contaminated withmycoplasma bacteria (3). When these cell lines are used in clinical settings, the effects can be disastrous. A single species, Mycoplasma pneumoniae, causes an estimated average of two million cases of bacterial pneumonia (4) and 100,000 hospitalizations each year (5). As gene therapies become more widespread, advanced testing methods will become ever more essential to ensure patients receive quality therapeutics to treat their conditions.

Mycoplasma species are elusive
 

Mycoplasma is everywhere, from animal-derived cell culture media to lab technicians’ hands and clothing (6). In addition to this prevalence, several characteristics make mycoplasma incredibly adept at evading detection.

First, mycoplasma are among the smallest self-replicating bacteria – only 2–3 µm across – which means they can pass through many filters and are undetectable using standard light microscopy. Second, mycoplasma are so small that they can appear in cell culture supernatant in concentrations of up to 10⁷ cells per mL without affecting the cells’ appearance (3). 

Finally, mycoplasma bacteria are Gram negative, making them resistant to the beta-lactam-based antibiotics commonly used to maintain cell lines. Taken together, these characteristics mean mycoplasma bacteria often end up in cell cultures undetected. 

Current detection methods are insufficient
 

Traditional mycoplasma detection methods include monitoring colony growth with broth or agar, staining or labeling nucleic acids, or testing for bacterial gene products. However, these tests are slow, taking up to four weeks to deliver results – which can severely delay manufacturing at crucial times (7)(8).

Some scientists have turned to quantitative PCR (qPCR) testing, which delivers results in just one day, as an alternative. However, qPCR measurements require calibration with a standard curve, giving a relative answer rather than an absolute one. Because it simply amplifies and measures DNA, the technique also cannot distinguish living mycoplasma from free-floating fragments in solution. To make this distinction, technicians must measure the ratio of genome copies (GCs) to colony-forming units (CFUs) – a ratio that unfortunately varies between cultures due to variable growth rates and culture conditions. Therefore, researchers must obtain an absolute count to accurately determine the ratio – a capability that qPCR lacks.

Absolute mycoplasma measurement
 

Unlike other detection methods, Droplet Digital PCR (ddPCR) directly quantifies nucleic acids, including mycoplasma DNA, using a water-oil emulsification technology to partition samples containing 10 µL of nucleic acid into 20,000 uniform droplets, each containing just one or a few gene copies. Then, the nucleic acids in each droplet are amplified using a probe that targets a genetic sequence unique to mycoplasma. Droplets containing the target sequence cleave the probe and emit a strong fluorescent signal upon amplification, whereas those without the target strand emit only a weak signal. The droplets are then streamed through a reader that counts fluorescent and non-fluorescent droplets so that the software can calculate the concentration of mycoplasma DNA in the sample using Poisson statistics. Because gene copies are counted in individual droplets, this process delivers absolute quantification.

Studies have already demonstrated ddPCR’s superior sensitivity and specificity relative to qPCR. For example, in one study, researchers used both technologies to examine three highly potent bacteria and found that qPCR overestimated the quantity of each species by a factor of two (9). In another, researchers examined whether ddPCR could accurately quantify three different mycoplasmas, a broad range representing various species found in nature: Acholeplasma laidlawii, M. pneumoniae, and M. hyorhinis. They found the limit of detection was low: 4.19GC/well, 6.29GC/well, and 5.63GC/well, respectively (10). The same group also examined their assay for cross-reactivity to assess its tendency to produce false positives; tests with three control species (Clostridium sporogenes, Lactobacillus acidophilus, and Streptococcus bovis) confirmed that ddPCR only detected the mycoplasma species of interest (10).

Quality control for the future
 

The future of gene therapy development depends on assuring quality control. As gene therapy becomes increasingly widespread, pathologists’ role will become even more vital. Central to AAV-mediated gene therapy quality control is the use of rapid, sensitive, and accurate technologies to assure confidence in the results. Good quality control will allow more patients than ever to benefit from the potential of these life-saving therapeutics.