Optimization and evaluation of an in vitro siRNA transfection protocol for preclinical toxicity screening in rat cortical cultures

Abstract

Small interfering RNAs (siRNAs) are a rapidly growing class of oligonucleotide therapeutics that modulate gene expression by inducing targeted mRNA degradation. siRNAs are potent and their effect is long-lasting, making them promising candidates for chronic neurodegenerative disease. However, siRNAs can elicit both on- and off-target toxicity, and while acute neurotoxicity has been characterized, long-term toxic effects remain poorly studied despite their relevance for chronic treatment. This study aims to optimize a protocol for in vitro screening of long-term siRNA toxicity (6 days to 3 weeks), to enable earlier triage of neurotoxic drug candidates, in line with the 3R principle of replacement, reduction, and refinement of animal testing. Fluorescently tagged siRNA was transfected into cortical neuron-astrocyte cultures from E17.5 rats using Lipofectamine RNAiMAX at varying siRNA concentrations, RNAiMAX dilutions, and complexing times. Cell health was evaluated using immunocytochemistry. Staining was done for morphological markers MAP2 and NeuN for neurons, GFAP for astrocytes, as well as all cell nuclei. The optimized transfection condition was used to evaluate a panel of siRNAs with known in vivo toxicity profiles. This revealed that the optimized method could identify siRNAs exhibiting substantial toxicity, while detection of milder in vivo toxic effects was more limited. Limitations in detecting mildly toxic effects may stem from low transfection efficiency in neurons, overshadowing by transfection-related toxicity or the lack of the immunoreactive cell type microglia in the cultures. These findings provide a foundation for in vitro long-term siRNA neurotoxicity screening.

Popular Abstract

Silence brain disease at its source – catch hidden toxicity in a dish and spare lab animals. siRNA drugs the hold promise of silencing diseases like Alzheimer's at the source. By screening drug candidates in a dish only a fraction of the animals is needed to filter out toxic candidates early. All new drugs have to be safety tested before they can hit the market, and often this safety testing comes at the expense of countless laboratory animals. As millions of people struggle with neurodegenerative diseases such as Alzheimer’s and Parkinson’s many of us will agree that this sacrifice is a necessary evil to deliver new life-saving therapies. Still, scientists work hard to replace, reduce, and refine the use of laboratory animals, a principle known as the 3Rs. To this end, a method was developed to test small interfering RNA (siRNA) drugs using rodent cells rather than live animals. Using cells in a dish to sort out the good seeds from the bad ones in terms of drug candidates is an established strategy when it comes to efficient drug design. A relatively new and very promising drug class for battling neurodegenerative diseases is siRNA therapies, but these have been difficult to screen in a petri dish because of the high sensitivity of brain cells and the characteristics of the drug molecule. Getting the screening method just right is therefore essential. If the method is well-optimized, one animal could give rise to hundreds of samples, instead of just one. Previous studies on siRNA drugs in rodents have encountered issues with hidden toxicity not appearing until weeks into testing. Since neurodegenerative diseases are chronic and treatments are meant to act during a long period of time, this kind of late-hitting toxicity is especially dangerous. This is one of the great advantages with cells-in-a-dish testing, as the experiment is faster to conduct than raising hundreds of rats. The power of siRNA therapy lies in its mechanism of action. If our genetic code is the blueprint for a house, then the engineer translates this into work orders (similarly to how DNA is transcribed to RNA) and the builders build the construction from these orders (like how RNA is translated into a protein). Many diseases are caused by overproduction or faulty construction of certain proteins, but siRNAs can intercept the building orders already at the mRNA level, hindering construction of new unwanted proteins. To do this, siRNAs need to enter the cells by crossing over the cell surface. This is difficult because siRNAs and cell surfaces are repulsed by one another, like two negative magnet poles. To overcome this challenge, the siRNA can be disguised using a positively charged delivery agent, which coats the siRNA, allowing it to enter the cell. The developed method successfully detected siRNAs of known high toxicity by detecting changes in the cells' appearance under the microscope. Finding siRNAs of milder toxicity proved more difficult, but the method still makes it possible to eliminate highly toxic siRNAs before toxicity testing goes forward to using living animals. This leads to faster and easier drug development, as well as fewer animals being exposed to dangerous drug candidates.

• author: Alexandersson, Linnéa and Envall, Tora
• supervisor: Kathrin Zeller; Christina Nodin
• organization: Department of Immunotechnology
• course: KIMM05 20261
• year: 2026
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• keywords: siRNA, transfection, RNAiMAX, primary neurons, primary astrocytes, rat cortex, in vitro, immunocytochemistry, electrical field stimulation, qPCR, HCA, neurotoxicity, method development, long-term toxicity
• language: English
• id: 9240444
• date added to LUP: 2026-06-17 16:33:55
• date last changed: 2026-06-17 16:33:55