Using RNA Interference to Affect Gene Expression and Cure Disease

By Alex Bui

Edited By: Katherine Hill, Nick Minor, Sienna Schaeffer, and Reed Owens-Kurtz

Credit: Vossman/Wikimedia Commons

For decades, many drugs acted by finding problematic proteins and then preventing them from functioning. For example, to treat breast cancers that require estrogen to stay cancerous, Anastrozole inhibits the enzyme that makes estrogen. Allergy medicines also use protein inhibition to stop surface proteins from recognizing inflammatory molecules. But what if you could stop those proteins from ever being made? Gene therapy is one approach for solving such problems. However, gene therapy has hit many obstacles on the way to becoming a mainstream treatment. RNA interference — one form of gene therapy — opens up new possibilities for treating diseases that were once thought to be untreatable and for improving existing methods.

To understand how RNAi works, it is first necessary to understand what DNA actually does. DNA is made of small molecules called nucleotides that contain one out of four nitrogen compounds to write a code for protein production. Although DNA holds all of the information necessary to create proteins, it needs to be rewritten as mRNA before it can be translated to protein that can be useful to the cell. If the protein product of a gene is not made, the gene is not expressed. In the simplest model of gene expression, DNA in cells is transcribed as mRNA which is translated by ribosomes to build proteins. RNAi is a phenomenon that was discovered fairly recently. Using RNAi, short RNA strands that don’t encode for proteins can either destroy mRNA or prevent it from being transcribed in the first place. In both cases, the end result is the same; the gene is deactivated and no protein is created.

RNAi acts on specific mRNA by using short guide strands that bind to the bases in the target RNA. With an eight nucleotide long region called the seed sequence, the guide strand must have near perfect binding to its target strand in order for the RNAi strand to affect gene expression. The guide strand’s level of complementarity outside the seed sequence determines whether the mRNA is immediately cleaved or sent to a special location in the cell to be destroyed. The production of the guide strands and their actions on the mRNA are broken up into the three categories of RNA interference.

(Lam et al., 2015)

In the first category, named “miRNA”, cells produce short RNA sequences from the genome that form a hairpin loop by binding the bases in a region on one end of the strand to the opposite end. Several proteins then cut away the unpaired region and shorten the double strand to about 20 nucleotides. A protein named AGO2 then selects most stable strand of the two and degrades it. The less stable RNA fragment then becomes the guide strand and associates with the AGO2 protein to form a complex that can now act on the target mRNA that needs to be destroyed. The region outside the seed sequence of miRNA is usually not very complementary to the target mRNA, so the target mRNA is brought to a special location in the cell where it is degraded.

The second form of RNAi is called siRNA. SiRNA likely originated as a defense mechanism against viruses and foreign DNA elements. Foreign double stranded nucleic acids are chopped into small segments like the miRNAs and are conjugated to AGO2. Unlike miRNAs, siRNAs have complete complementarity to the target mRNA because they are made from strands identical to the target sequence. Complete complementarity causes the guide strand complex to immediately cleave the target mRNA.

The last form of RNAi is piRNA. piRNA is only active in germ cells and is used to keep the genome unchanged. Transposons, also known as “jumping genes”, produce mRNA that inserts the transposon sequence into another place in the genome. This can have serious effects if the transposon is inserted into important genes or promoting elements. Upon binding, piRNA goes to the nucleus of the cell and covers up the DNA that encoded the transposon so that it can’t be expressed in the future.

Manipulating these forms of RNAi has wide-ranging potential to treat diseases with gene therapy and to innovate treatments for modern diseases. Recently, RNAi treatments have received FDA approval and are progressing to late stage clinical trials to prove safety and efficacy in humans.

Cholesterol Molecule (Image Credit: Wikimedia Commons)

One such treatment is Inclisiran, which aims to lower cholesterol levels and is currently undergoing clinical trials. Heart disease is the leading cause of death in the United States and is often tied to high cholesterol. Traditionally, statins were the default treatment for those that suffer from high cholesterol. Statins work to lower blood cholesterol levels by inhibiting the proteins that produce cholesterol. However, their side-effects are wide-ranging and may include an increased risk of developing diabetes (Sattar et al.,2010). For this reason, developing new treatments with fewer serious side effects is an important issue as the prevalence of diabetes continually increases.

Inclisiran’s mechanism of action is not quite as simple as statins’. Inclisiran is an artificial siRNA that targets the mRNA of a protein called PCSK9. PCSK9 marks cholesterol receptors for degradation. This prevents cells from pulling cholesterol out of the bloodstream. By stopping the production of PCSK9, the researchers hoped to see lower average cholesterol levels in patients that received treatment. To test this, 501 individuals at high risk of developing heart disease were placed into one of eight experimental groups. Two of these groups were control groups and received placebo injections. The other six received varying levels of Inclisiran at single or double doses and were periodically screened for cholesterol levels over the course of 240 days. The results of the experiment convincingly showed that Inclisiran treatment lowered blood cholesterol in patients compared to patients that received placebo treatment.

(Ray, 2017)

The study also suggested that Inclisiran treatment can be both safe and effective (Ray, 2017). Side effects of treatment were seen in both placebo and Inclisiran injections at all dosage strengths. These side effects were generally mild to moderate in terms of severity and were not specific to any particular level of treatment. Two deaths occurred in the study but appeared to be unrelated to the treatment. At this point, it is too early to conclude that Inclisiran treatment doesn’t have any serious side effects, but hopefully Inclisiran proves to be a safe and effective treatment.

RNAi treatments have also seen success in treating spinal muscular atrophy and preventing vision deterioration due to blood vessel growth. One example is Spinraza, which was approved in late 2016 to treat spinal muscular atrophy by correcting a mRNA processing error. Macugen treats eye degeneration by inhibiting the production of vascular endothelial growth factor which causes growth and spread of blood vessels.

RNAi currently is also undergoing research as a treatment of cancer. Many cancers are caused by overexpression of cellular receptors or protein products. RNAi may be a way to regulate expression of these genes to normal levels and stop the proliferation of tumors. Diseases such as ovarian cancer, prostate cancer, and thyroid cancer have had progress made in developing a cure and hopefully can become a widespread treatment for many cancers. RNAi’s ability to fine tune the expression of cancer causing protein products shows promise to become a widespread method of treatment that may be less burdensome than radiation or chemotherapy. The field is still developing and needs more research to understand RNAi and utilize it to its full potential.


  1. Lam, J. K., Chow, M. Y., Zhang, Y., & Leung, S. W. (2015). SiRNA Versus miRNA as Therapeutics for Gene Silencing. Molecular Therapy — Nucleic Acids, 4. doi:10.1038/mtna.2015.23
  2. Sattar, Naveed et al. Statins and risk of incident diabetes: a collaborative meta-analysis of randomised statin trials. The Lancet , Volume 375 , Issue 9716 , 735–742
  3. Ray, K. K., Landmesser, U., Leiter, L. A., Kallend, D., Dufour, R., Karakas, M., . . . Kastelein, J. J. (2017). Inclisiran in Patients at High Cardiovascular Risk with Elevated LDL Cholesterol. New England Journal of Medicine, 376(15), 1430–1440. doi:10.1056/nejmoa1615758