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A by pharmaceutical researchers from Utrecht, focusing on the targeted delivery of the "DNA scissors" CRISPR-Cas9, is garnering significant attention from peers. Since its publication in February this year, it has already been cited relatively often in scientific publications. The reason? Not the successful experiment that is also discussed in the paper, but the other experiment that resulted in an unexpected and fatal outcome for the laboratory animals. Group leader Enrico Mastrobattista: “I am glad that these negative results are finally shared, as it helps prevent duplication of work and unnecessary suffering in laboratory animals.”

Mastrobattista and his team study how tiny spheres composed of lipids, known as lipid nanoparticles, can be used to deliver drugs precisely where they are needed in the body. These lipid nanoparticles can also serve as carriers to deliver CRISPR-Cas9 to the desired location.

DNA scissors

CRISPR-Cas9 originally is a bacterial defence mechanism that allows bacteria to neutralize viruses. It functions like a pair of molecular scissors, cutting DNA at specific locations. Cas9 is the enzyme responsible for making the actual cut, while a short RNA molecule bound to the Cas9 enzyme, the guide RNA, determines the exact location of the cut.

It is also important to publish results when something does not work, as it helps prevent the unnecessary duplication of effort and saves time and money. Moreover, it prevents a lot of suffering in laboratory animals.

CRISPR-Cas9 technology enables precise modification of DNA in living organisms, making it a groundbreaking tool in medicine and other fields. For instance, it holds the potential to cure genetic diseases by identifying and deactivating specific genes that produce harmful proteins. Furthermore, it even allows for the direct editing of DNA sequences at targeted locations, making it possible to correct mutated genes responsible for certain diseases.

RNA or protein

The technique is not flawless. The Cas9 enzyme occasionally cuts DNA at unintended sites. These undesired cuts, also called off-target effects, must be minimized. This can be achieved by optimizing the guide RNA to improve precision, but also by keeping the residence time of the Cas9 in the cell as short as possible and allowing it to be active only in those cells where it is needed.

It motivated me as a young scientist to realize that negative results can still be educational.

The latter can potentially be achieved using nanoparticles. Here, the specific contents of the nanoparticles play a crucial role in determining how long CRISPR-Cas9 remains active. Mastrobattista: "There are two ways to deliver CRISPR-Cas9: through proteins or through messenger RNA (mRNA). When using mRNA, the activity lasts about five to seven days, while with protein, it lasts only for a few hours. So in principal, protein is much safer, as it lowers the risk of off-target effects."

Strong immune response

In the study currently attracting significant attention, researcher Johanna Walther investigated how effectively CRISPR-Cas9 can silence a specific gene in the liver of mice. These types of complex experiments, which examine how nanomedicines are transported and taken up by cells, can really only be conducted in laboratory animals. This is because current animal-free model systems are still not sufficiently advanced. In her work, Mastrobattista's former PhD candidate compared the effects of a treatment with lipid nanoparticles filled with protein versus the effects of a treatment with nanoparticles filled with mRNA.

The protein-filled nanoparticles were not found to be safer. On the contrary. Mastrobattista: "The mice that received the protein treatment all completely unexpectedly died within two days. We still need to determine the exact cause, but it appears that the immune system was strongly activated, leading the mice into septic shock."

Publication bias

The study also yielded positive results: the mRNA-filled nanoparticles were effective, successfully turning off the targeted gene in sixty percent of liver cells. Moreover, all the mRNA-treated mice survived, with no negative health effects observed. Mastrobattista: "Others have reported similar positive results before. From my point of view, the reason this article has gained so much attention is because the negative results are finally being shared."

Mastrobattista says that he recently presented the results at a conference, where several attendees approached him to share that they had encountered similar negative results in their own experiments—but no one had published those findings. "This illustrates the so-called publication bias, where only successful experiments are shared," he explains. "However, it is also important to publish results when something does not work, as it helps prevent the unnecessary duplication of effort and saves time and money. Moreover, it prevents a lot of suffering in laboratory animals."

We see potential solutions for working with protein, which is desirable because it is potentially safer.

Negative results

Publication bias occurs when researchers themselves do not share their negative results, or when journals deem these results unsuitable for publication. Mastrobattista knows why the negative results were published this time: “We also had positive results, which gave confidence that the experiment was done well.”

Johanna Walther currently works as project leader at the Netherlands Cancer Institute. Walther: “I have always appreciated that Enrico is a supervisor who encourages the publication of negative data. It also motivated me as a young scientist to realize that negative results can still be educational. The death of the mice was grim, but it also conveyed a clear message that needed to be shared.”

Further research

Despite the results, Mastrobattista remains undeterred. “CRISPR-Cas9 is of bacterial origin, so it is natural for the immune system to react to it,” he says. “We see potential solutions for working with protein, which is desirable because it is potentially safer. Perhaps the protein we used was not completely pure, which may have triggered a strong immune response. We could also modify the protein to reduce the immune reaction while ensuring it still functions effectively.”