shRNA: Solving the Subtle

A report published in the Proceedings of the National Academy of Sciences illustrates how subtle structural differences between two types of RNAs-artificial small hairpin RNA and endogenous microRNA-may help explain the difference between shRNA having a therapeutic effect or being toxic.1

The researchers found that designing miRNA-like shRNAs offered a potential solution to shRNA toxicity. But industry researchers approached by SciBX also emphasized the need for a solid initial shRNA design process to avoid unwanted side effects in the first place.

shRNAs consist of a double-stranded stretch of RNA (21 nucleotides in length) capped off on one end by a single-stranded RNA loop (6-8 nucleotides long). The active moiety, the 21-nucleotide guide strand from the double-stranded portion of the shRNA, is complementary to a target mRNA. The remaining strand is called the passenger strand.

shRNA molecules can be expressed from transgenic or viral vectors and can also be introduced into cells exogenously. Once inside the cytoplasm, an shRNA is split by the cellular machinery into the single-stranded guide and passenger strands. The guide strand proceeds to form an RNA-protein complex called the RNA-induced silencing complex (RISC), which cleaves its complementary mRNA. The result is reduced expression of the corresponding protein (see "Staking out the RNAi landscape").

In the PNAS study, a team led by Beverly Davidson, professor of neurology at the University of Iowa, assessed the feasibility of using shRNA to treat Huntington's disease in mice expressing the mutant form of huntingtin that is responsible for the disease. The researchers tested various shRNA constructs delivered by adenoassociated viral (AAV) vectors and determined changes in levels of the toxic protein.

The researchers began by testing a panel of 35 shRNA constructs that target different parts of the huntingtin gene, looking for reductions in huntingtin mRNA levels in mouse and human cell cultures. The three best candidates were injected into the brains of transgenic mice with HD. Although all three reduced huntingtin mRNA levels, two of them also triggered astroglial inflammation and striatal toxicity.

"We set out to test the hypothesis of whether or not we could reduce huntingtin expression in the adult brain," Davidson told SciBX.

Read the full 3493 word article

User Sign In

Article Purchase

This article may not be distributed to non-subscribers

PURCHASE THIS ARTICLE FOR LIMITED ONE-TIME DISTRIBUTION AND WEBSITE POSTING $995.00 USD

PURCHASE