RNA-based drugs have been slow to realize their therapeutic promise because they often find themselves trapped in endosomes and delivered to degradative lysosomes. Yet if RNA-based drugs could be broken out of endosomes, they could complete their mission, the downregulation of pathogenic genes.

To help RNA-based drugs exit the endosome and enter the cytosol, researchers at Lund University in Sweden tried the molecular equivalent of assisting a jailbreak. They used chloroquine and other small molecules to disrupt endosomal membranes. Then, to gauge the effect of these molecules, the scientists looked for signs of endosomal damage in tumor spheroids. Ultimately, using live-cell microscopy, the scientists established that chloroquine freed a small interfering RNA (siRNA), enabling the siRNA’s knockdown of a targeted gene.

Encouraged by this result, the scientists asserted that their imaging technique “will be widely applicable, facilitating efforts to improve delivery of siRNA and other nucleic acid-based therapeutics.” These words appeared in a paper (“Imaging small molecule-induced endosomal escape of siRNA”) published April 14 in Nature Communications.

The scientists, led by cancer researcher and physician Anders Wittrup, used a live-cell microscopy technique that incorporated cytosolic galectin-9 as a sensor of membrane damage. They found that membrane-destabilizing drugs are diverse and target multiple intracellular compartments. They also observed, however, that mismatch between siRNA-containing and drug-targeted compartments limits the siRNA activity improvement.

“The completely new microscopy methods make it possible to study in detail when lysosomes and other structures in the cell are opened up for RNA delivery,” said doctoral student Hampus Du Rietz, the lead author of the current study. “This is a technique that is in high demand in both academic research and within the pharmaceutical industry.”

“We have shown how chloroquine can be used so that the siRNA molecules enter the cytosol, that is, the space between the cell membrane and the nucleus of the cell,” added Wittrup.

Membrane damage caused by chloroquine in spheroids incorporating cervical cancer cells. The spheroid on the right has been treated with the substance chloroquine, which resulted in widespread membrane damage in the cells. This damage is seen as orange spots. The tumor cell nuclei are blue. [Hampus Du Rietz, Lund University]

This way of opening the lysosomes, which previously encapsulated the RNA molecules and thus prevented their effect, means that a fundamental obstacle for the use of siRNA- and other RNA-based drugs is now potentially on the way to being removed.

“We demonstrate efficient release of ligand-conjugated siRNA from vesicles damaged by small molecules, enhancing target knockdown up to ∼47-fold in tumor cells,” the authors of the current study wrote. “We also show widespread endosomal damage in macroscopic tumor spheroids after small molecule treatment, substantially improving siRNA delivery and knockdown throughout the spheroid.”

The researchers emphasized that their work with tumor spheroids could facilitate the optimization of the endosomal disruption strategy, an approach that could boost the intracellular delivery of siRNA- and other RNA-based drugs. (In the current study, the researchers studied siRNA molecules linked with cholesterol, which means that they are taken up effectively by most tumor cells.)

Going forward, the researchers hope to help siRNA realize its promise, which began to be appreciated in the 1990s, when it was discovered that siRNA could be used to turn off virtually any gene. The phenomenon was named RNA interference. In 2006, the discovery was awarded the Nobel Prize in Physiology or Medicine. There were considerable hopes that RNA interference could be used in the treatment of virus infections, cancer, and other diseases.

“Two siRNA drugs have been approved by the FDA,” Wittrup noted, “but as yet, no drug has been approved for clinical use in cancer.”

One considerable advantage of using RNA molecules is that they can be rapidly developed and produced. The major problem for all sorts of RNA drugs is getting these molecules into the interior of the cell, the so-called cytosol, where they have an effect. The size of the siRNA molecule—about 50 times larger than a typical drug molecule—is a factor.

“Even if we can make tumor cells take up siRNA, it has been observed that 99% appears to get caught in a type of cellular waste dump, the so-called lysosome,” explained Wittrup.

“Our findings open up exciting possibilities for how RNA can reach into the interior of cells,” he concluded. “I think it will play a crucial role in developing RNA drugs for the treatment of diseases for which we currently lack effective drugs.”

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