Targeting siRNA to Microglia to Suppress PU.1 Expression and Reduce Neuroinflammation – Fight Aging!

Researchers here report on their development of a means to target microglia in the brain with small interfering RNA (siRNA) to reduce PU.1 protein expression. PU.1 is implicated in the regulation of inflammation in microglia, and a number of groups are attempting to produce a basis for therapies. Chronic inflammation driven by microglia is a feature of aging and neurodegenerative conditions. Unresolved, constant inflammation is disruptive of tissue structure and function, and the brain is no exception. Inflammation is thought to be an important factor in the onset and progression of the most common forms of neurodegeneration, including Alzheimer’s disease.

In a prior study researchers showed that blocking the consequences of PU.1 protein activity helps to reduce Alzheimer’s disease-related neuroinflammation and pathology. The simplest way to test whether siRNA could therapeutically suppress PU.1 expression in microglia would have been to make use of an already available delivery device, but one of the first discoveries in the study is that none of eight commercially available reagents could safely and effectively transfect cultured human microglia-like cells in the lab.

Instead the team had to optimize a lipid nanoparticle (LNP) to do the job. LNPs have four main components and by changing the structures of two of them, and by varying the ratio of lipids to RNA, the researchers were able to come up with seven formulations to try. Among the seven candidates, one the team named “MG-LNP” stood out for its especially high delivery efficiency and safety of a test RNA cargo. What works in a dish sometimes doesn’t work in a living organism, so the team next tested their LNP formulations’ effectiveness and safety in mice. Testing two different methods of injection, into the body or into the cerebrospinal fluid (CSF), they found that injection into the CSF ensured much greater efficacy in targeting microglia without affecting cells in other organs. Among the seven formulations, MG-LNP again proved the most effective at transfecting microglia.

Once they knew MG-LNP could deliver a test cargo to microglia both in human cell cultures and mice, the scientists then tested whether using it to deliver a PU.1-suppressing siRNA could reduce inflammation in microglia. In the cell cultures, a relatively low dose achieved a 42 percent reduction of PU.1 expression (which is good because microglia need at least some PU.1 to live). Indeed MG-LNP transfection did not cause the cells any harm. It also significantly reduced the transcription of the genes that PU.1 expression increases in microglia, indicating that it can reduce multiple inflammatory markers.

The final set of tests evaluated MG-LNP’s performance delivering the siRNA in two mouse models of inflammation in the brain. In one, mice were exposed to LPS, a molecule that simulates infection and stimulates a systemic inflammation response. In the other model, mice exhibit severe neurodegeneration and inflammation when an enzyme called CDK5 becomes hyperactivated by a protein called p25. In both models, injection of MG-LNPs carrying the anti-PU.1 siRNA reduced expression of PU.1 and inflammatory markers, much like in the cultured human cells.

Link: https://picower.mit.edu/news/nanoparticle-delivered-rna-reduces-neuroinflammation-lab-tests