Safety and our project
The most relevant safety aspect of our project, aside from lab safety during experimental work, was the safety of our product. The goal was to develop a fast and accurate point of care test which would facilitate treatment of Schistosomiasis as well as contribute to stopping the spread of the disease in affected countries. With this purpose in mind, the safety aspects concern mainly the use in a local perspective and in the accuracy of the test results.
Biosafety
To make the product safe in a biosafety perspective, the first aspect we considered was the choice of chassis for our product. We chose to use a strain of Saccharomyces cerevisiae, originally derived from the widely used laboratory strain S288C, according to the following table [1].
| Strain | Parental Strain | Specification |
|---|---|---|
| BY4741 | S288C | MAT(α) his3 Δ1 leu2 Δ0 met15 Δ0 ura3 Δ0 |
| yWS677 | BY4741 | sst2Δ0 far1Δ0 bar1Δ0 ste2Δ0 ste12Δ0 gpa1Δ0 ste3Δ0 mf(α)1Δ0 mf(α)2Δ0 mf(α)1Δ0 mf(α)2Δ0 gpr1Δ0 gpa2Δ0 |
| Design 4 (from Tom Ellis lab) |
yWS677 | ura3Δ0:pCCW12-STE2-tSSA1-pPGK1-GPA1-tENO2-pRAD27-LexA-PRD-tENO1-URA3; leu2Δ0: LexO(6x)-pLEU2m-sfGFP-tTDH1-LEU2 |
| Design 4 ura3Δ | Design 4 (S288C) | ura3Δ0 |
According to table 1, Design 4 strain has no hazardous properties and should be safe to use for humans and not pose risks to the environment even in the case of accidental release. The strain is also equipped with the selection marker URA3, which is not only necessary to select for the strain, but also provides an additional safety measure in case of accidental release. This is important when considering that the goal is to use this cell based system as a PoC test. In addition, the selection does not require any form of antibiotics. This is a simple but important detail to avoid contributing to antibiotic resistance.
It is crucial to be able to ensure safety when bringing GMO outside of the lab considering that GMO is strictly regulated in many regions and a subject that needs to be thoroughly evaluated in order to minimize risks which can be difficult to predict.
Patient Safety
If the test gives many false positive results, this could result in the patient receiving unnecessary treatment, thus it is very important that the test is accurate to ensure patient safety. A few solutions were implemented to assure this, the most relevant being the design of a specific DNA detection system consisting of a zinc-finger complex. To evaluate if this system meets our expectations, we also compared the robustness and target DNA-affinity of our zinc-fingers with the synTALE, which is used by our partner team Utokyo with the same purpose of binding strongly and specifically to a target DNA sequence.
WWe also wanted to implement a set of controls in the test that would, in the case of a false positive or negative result, discover them directly by looking at the controls. More about this control setup can be found on the implementation page.
Another aspect of patient safety is the choice of sample to use for the diagnosis of the patient. The free parasitic DNA can be found in serum, urine and saliva [2]. We chose to use saliva to minimize the required invasiveness of the sampling technique. This can be compared to the procedure that is required when obtaining a blood sample, which requires sterile equipment and trained staff, thus posing a higher risk to the patient's safety while also being less convenient for a PoC test. The same is true for taking a urine sample, which does not cause major risks to the patient, but the question of patient comfort and integrity is instead more relevant.
Lab safety
When considering the lab safety and our personal safety during the development of our product, we have carefully followed local regulations and guidelines. This includes writing risk assessments and safety declarations prior to the experiments, as well as participating in the required safety presentation at our lab. During every experiment we also used suitable safety equipment like appropriate gloves, lab coat and glasses, depending on the level of risk that was associated with the particular experiment.
To minimize the risk of contamination, proper sterile techniques were practiced. This includes keeping workspaces sterile using LAF-benches and bunsen burners and practicing correct waste management. We also always had close contact with our supervisors and responsible lab engineers, who were happy to answer all of our safety related questions.
References
[1] Mortimer RK, Jhonston JR. GENEALOGY OF PRINCIPAL STRAINS OF THE YEAST GENETIC STOCK CENTER. Genetics. 1986May1;113(1):35–43.[2] Weerakoon KG, McManus DP. Cell-free DNA as a diagnostic tool for human parasitic infections. Trends in Parasitology. 2016Feb1;32(5):378–91.