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Researchers Name Endosomal Pathway as Source of Difficulties for RNAi in Primary Cells

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As RNAi’s popularity as a research tool continues to grow, scientists still face problems with delivering siRNAs to primary cells, especially primary neurons. But research coming out of the University of Gottingen in Germany, and published recently in Biochemical and Biophysical Research Communications, indicates that a solution might exist in the circumvention of the endosomal pathway.

“One of the major constraints in RNAi studies employing primary mammalian cells is the efficient and non-toxic transfection of siRNA,” Paul Lingor and colleagues from the University of Gottingen wrote in the article.

Although a recent report by Harvard Medical School’s Kenneth Kosik in Proceedings of the National Academy of Sciences showed that RNAi can function in principle in cultured primary mammalian neurons, the German researchers wrote, “survival of cells in dissociated neuron cultures was markedly impaired by cationic lipid toxicity.

“Compared to most other primary cells, primary neurons are known to be even more susceptible to liposome toxicity,” they wrote. “Our present study, therefore, evaluates the possibility of siRNA delivery … without the use of cationic lipids or other transfection reagents.”

The researchers wrote that they applied naked Cy3-labeled siRNAs to primary hippocampal neuron cultures, finding that the siRNAs become visible after several days. Co-localization with endosomal/lysomal antigens revealed primary uptake of the siRNAs into the endosomal compartment.

“Cy3 staining was also found in the cytoplasm, indicating that Cy3-siRNA partially is released from endo-/lysomal vesicles,” Lingor et al. wrote. “In contrast, the nucleus of the cells remained completely spared.”

Further analyses indicated that siRNAs adhere to dissociated primary cells during the preparation procedure in amounts that would be sufficient for RNAi induction, they stated. “We thus expected that application of naked siRNA will induce silencing effects in primary neuron culture. Nevertheless, no convincing silencing effect … could be observed using siRNA concentrations up to 250nm.”

Additionally, “primary neuron cultures treated with naked siRNA remained morphologically unchanged, yet showed a moderate impairment of overall culture metabolism in metabolic tests.”

The researchers suggested a number of possibilities for the non-specific effects on cell metabolism, including toxicity of the siRNAs themselves due to modification within the endosomic environment as a result of low pH values and high nuclease activity.

“Our results suggest that the uptake of siRNA in the endosomal compartment represents the limiting step in siRNA transfection into primary neurons,” the paper’s authors concluded. “Synthetic siRNA delivery agents circumventing the endosomal pathway would thus be the method of choice for this application.”

While cell-penetrating peptides have been proposed as a means to enter a cell in a way other than the endosomal pathway, they noted, the use of these peptides still lacks supporting evidence.

“We are currently examining the possibility of complexing siRNAs to protein transduction domains,” they wrote. “Potential candidates might include the HIV-1 Tat, the HSV-1 VP22, or the Drosophila Antennapedia homeoprotein.”

Cationic lipid-based agents are able to release complexed nucleic acids from the endosome and target siRNAs to their functional localization, they added. But in order to use these lipids, the development of less cytotoxic formulations is necessary.

—DM