Written by Frida Niss Edited by Dr. Hayley McLoughlin

One drug to treat them all: an approach using RNA interference to selectively lower the amount of mutant protein in all polyglutamine diseases. Work by a group in Poland shows initial success in Huntington’s Disease, DRPLA, SCA3/MJD, and SCA7 patient cells.

Can one drug treat nine heritable and fatal disorders? Polyglutamine diseases are disorders in which a gene encoding a specific protein is expanded to include a long CAG repeat. This results in the protein having a long chain of the amino acid glutamine, which disturbs the ability of the protein to fold itself and interact correctly with other proteins. This type of malfunctioning protein would normally be degraded by the cell, but in the case of polyglutamine proteins this seems unusually difficult. This causes a gradual build-up of faulty protein that disrupts several cellular pathways, eventually leading to cell death in sensitive cells. Currently there is only symptomatic treatment of these fatal diseases available, and they do not slow down the disease progression. One promising line of research is investigating the possibility of lowering the amount of these disease proteins using RNA interference.

RNA interference is the method by which a gene is silenced through a manipulation of a natural defense mechanism against viruses. When a virus attacks, it tries to inject DNA or RNA like particles to hijack the cell’s machinery for its own survival. To defend itself, the cell uses the RNA interference pathway, where the protein Dicer slices the DNA/RNA into smaller pieces and loads it into the RNA-induced silencing complex (RISC complex). The RISC complex finds all DNA/RNA particles in the cell with the same sequence and destroys them, effectively hamstringing the virus.

This machinery can be co-opted as a potential tool for treating neurodegenerative diseases caused by harmful mutant proteins. By inserting a small interfering RNA (siRNA), we can target the mRNA that codes for the harmful protein and trick the RISC complex into degrading it. In polyglutamine diseases, this has been successful when the mutant mRNA possesses a small mutation called a single nucleotide polymorphism (SNP). However, when an siRNA is delivered to a cell using a vector, which is a circular piece of DNA carrying genetic material, the Dicer protein tends to process the siRNA in unpredictable ways. This means that the treatment may not always be selective, and can end up targeting the normal protein as well. Moreover, not all patients have the same SNPs, so several drugs for every disease might be needed.

In the paper by Kotowska-Zimmer and colleagues they have used short hairpin RNAs (shRNAs) targeting the CAG repeat tract itself instead of siRNAs targeting SNPs around the CAG repeat tract. shRNAs fold themselves like a hairpin when transcribed, and this loads them into the RISC complex through a somewhat different pathway, with less degradation along the way than conventional siRNAs. The second part that is different to other RNA interference strategies in this study is that the shRNA does not completely match the CAG repeat, but contains mismatches. This means that the RISC complex cannot actually cut and degrade the mRNA, and ends up simply sitting on the CAG repeat tract instead. The longer the repeat tract, the more RISC complexes can fit on the tract and block translation. Using this type of RNA interference Kotowska-Zimmer and colleagues have tried to lower the expression of huntingtin, atrophin-1, ataxin-3 and ataxin-7 proteins in cellular models of the corresponding polyglutamine diseases.

They decided to try four versions of shRNAs that had very slight differences in sequence, introducing a mismatched nucleotide in one or two places. They first tested these four shRNAs by using cells that would give off light based on how much huntingtin protein was being produced. All four shRNAs caused the cells producing mutant huntingtin to give off less light than the cells producing normal huntingtin, but the efficacy and selectivity differed a lot between them. The most selective and efficient of the four shRNAs was also tested in three different cellular Huntington’s Disease (HD) models, using cells taken out of HD patients or HD mice. In all three cases, the mutant huntingtin protein was preferentially silenced.

This group hopes to design an shRNA that can decrease all polyglutamine disease proteins, not just huntingtin. Therefore, in the next step they tested their four shRNAs in a different disease model: Dentato-rubral pallidoluysian atrophy (DRPLA). DRPLA differs from HD in that it affects the entire central nervous system as opposed to mainly the striatum, but also in that the CAG tract is localized in the middle of the protein, as opposed to the very edge. In cells taken from DRPLA patients, the four shRNAs performed similarly to in the HD models: they selectively targeted the mutant mRNA and thus lowered the mutant protein containing the long glutamine tract. The same happened when they tried with patient cells taken from Spinocerebellar ataxia type 3 (SCA3) and Spinocerebellar ataxia type 7 (SCA7) patients, though the efficacy and selectivity of the shRNAs was lower in the SCA7 derived cells. Among all disease studies, the shRNA named A2R showed the most promise for being a universal polyglutamine protein lowering agent.

Since several important proteins not related to polyglutamine diseases can also contain longer CAG tracts, Kotowska-Zimmer and colleagues tested four such proteins using A2R, and confirmed that none of these were affected by the shRNA. However, one mRNA that fully matched the A2R shRNA was lowered in the cells tested, meaning that the protein made from that mRNA might be decreased by the treatment. This is called an “off target effect”. The consequences of this are difficult to predict, since the expression is highly specific to certain cells. So testing this A2R shRNA in a mouse model will be a critical next step. Animal model tests will need to be done before we can even think of testing this in humans.

Polyglutamine diseases are deadly, and even with a slow disease progression, the symptoms can be detrimental to the quality of life. Finding a preventative treatment to keep the expression of harmful polyglutamine proteins in check even before symptoms are developed is therefore a very attractive idea that has occupied many research teams around the world. If one single drug could treat all polyglutamine diseases to boot, that would be sensational. So far, many different variants of RNA interference drugs have been developed, and clinical trials are already running for some of them, with varying results. The variant developed by Kotowska-Zimmer and colleagues shows promise, and any future animal models and clinical trials will be exciting to follow.

Key Terms

RNA interference: A cellular defence mechanism that protects the cell from unfamiliar nucleic acids, and can be used to regulate its own expression. To accomplish this, several RNA particles can be used, for example

• Short hairpin RNAs: RNA molecules folded like a hairpin that can be used in the RNA interference pathway.
• Small interfering RNAs: Short double-stranded RNA molecules that can be used in the RNA interference pathway.

To learn more about how RNA  interference is being used by scientists to develop new treatments and therapies, you can read our Snapshot on RNAi.

RISC complex: The complex in charge of finding and degrading unwanted RNA in the RNA interference pathway.

Cellular disease models: Cells that express disease proteins and enable researchers to study molecular pathways involved in the pathology of the disease. Sometimes they can be biopsied from patients to reflect the disease more accurately.

Conflict of Interest Statement

The authors and editor declare no conflict of interest.

Citation of Article Reviewed

Kotowska-Zimmer et al. Universal RNAi Triggers for the Specific Inhibition of Mutant Huntingtin, Atrophin-1, Ataxin-3, and Ataxin-7 Expression. Mol Ther Nucleic Acids, 2020 March 6. (19) 562-571.  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957814/