How siRNA silences gene expression
First, a quick introduction to gene expression and gene silencing.
Gene expression is the process cells use to turn the instructions encoded in our DNA (genes) into functional proteins. A key intermediary in this process is messenger RNA (mRNA), which copies the instructions from the DNA in the cell nucleus, carrying the information to the cellular machinery outside the nucleus that manufactures proteins. Every protein-coding gene in our DNA has a corresponding mRNA that executes the gene’s expression.
While the development of COVID-19 vaccines has focused attention on mRNA as a therapeutic agent to prevent or treat diseases, mRNA has a lengthy history as a therapeutic target for siRNAs. siRNAs are short sequences of RNA that interfere with gene expression by binding an mRNA and promoting its degradation before it reaches the cell’s protein-making machinery, effectively ‘silencing’ expression of the corresponding gene. The Nobel prize-winning discovery of this naturally occurring mechanism, also known as RNA interference (RNAi), created the possibility of using synthetic siRNAs as therapies to reduce or eliminate the expression of a disease-related protein by targeting its corresponding mRNA.
siRNA for cardiovascular disease due to Lp(a)
Three of the four siRNA therapies approved since 2018 and many more siRNA therapies in development are intended to treat rare non-cardiovascular diseases – those caused by a mutation in a single gene that gives rise to single mutant mRNA and the corresponding disease-causing protein. The fourth siRNA therapy is approved to treat both heterozygous familial hypercholesterolemia (FH) – an inherited, rare disease involving elevated LDL-C levels – and atherosclerotic cardiovascular disease (ASCVD), a more common condition.
Common diseases that are candidates for siRNA therapies may be associated with gene variants that are not pathological mutations and therefore do not directly cause a disease. The LPA gene, whose mRNA is the target of our Lp(a)-lowering siRNA therapy, is a good example of this type of gene.
LPA encodes a protein called apolipoprotein(a), which is a component of Lp(a), a cholesterol-rich particle that is closely related to, but distinct from, LDL-C. Lp(a) is recognised as a major risk factor associated with increased risks of heart attack,2,3 aortic stenosis,4 heart failure,5 stroke6 and death.7,8
However, unlike LDL-C, Lp(a) levels are not significantly modifiable by lifestyle changes.9,10 Instead, Lp(a) levels are genetically determined by the individual’s LPA gene variant.11,12 As these variants are not pathological mutations, the person may not experience disease symptoms for years and may even be unaware of their elevated Lp(a) levels. Even so, the condition is common: one in five people worldwide has high levels of Lp(a), which is defined as 50 mg/dl or 120 nmol/L.13-15
Cholesterol-reducing medicines such as statins have no effect on Lp(a) and some can even increase Lp(a) levels; currently there are no approved Lp(a)‑reducing therapies. This leaves the 20 percent of the world’s population who have elevated Lp(a) in need of therapies that can reduce their risk of cardiovascular disease.
Developing an siRNA to silence LPA
Silence, among other companies, is developing an investigational siRNA therapy that is designed to silence LPA expression by targeting its mRNA.
LPA is an eminently suitable target for an siRNA therapy because silencing the gene is expected to be safe. Assessments of human genetic data have revealed that cardiovascular risk is the only phenotype associated with LPA gene variants and the resulting Lp(a) levels: high levels of Lp(a) are associated with high risk; low levels are associated with low risk. Some individuals have zero levels of Lp(a) and the only known phenotype they present is a much-reduced incidence of cardiovascular events. Some information suggests that very low levels could increase the incidence of diabetes, but this is controversial and the potential mechanism is unclear. This information indicates that silencing LPA with a properly designed siRNA therapy could reduce the risk of cardiovascular disease associated with elevated Lp(a), while minimising the potential for any unwanted or unexpected side effects.
This accords with Silence’s general approach to selecting targets for our siRNA therapies: we silence a gene that has little or no effect on phenotypes outside the disease, thereby maximising safety. While safety is important in treating any disease, it has particular importance in a common chronic disease, such as hyperlipidemia, where patients may live for decades before they experience any overt symptoms from their condition. Over this possibly long period of time, patients are not likely to tolerate a therapy with even minor side effects that interfere with their quality of life.
After selecting LPA mRNA as our therapeutic target, we applied in silico techniques to design the sequence of the siRNA, both to maximise its silencing effect on the mRNA and minimise its potential to bind other mRNAs which might produce unwanted side or off‑target effects. The ability to predict and screen out siRNA sequences that could cause side effects is possible due to the precise mechanism of nucleotide base pairing, also known as Watson-Crick base pairing, by which siRNAs silence their mRNA targets. As siRNAs are both precision medicine and targeted medicine, we anticipate our investigational therapy for elevated Lp(a) will have a wide safety margin.
We further enhanced safety by conjugating the siRNAs to the amino‑sugar molecule GalNAc, which enables highly specific delivery of siRNAs to the liver, where thousands of genes, including LPA, are expressed. By limiting siRNA uptake primarily to liver cells, we reduce or eliminate uptake by other tissues that do not express LPA.
siRNA dosing: better compliance, better outcomes
Another key advantage of siRNA therapies is that their effects are long‑lasting. Patients need only receive a few therapy injections each year and less frequent dosing should increase patient compliance with treatment compared with oral, small molecule drugs, most of which must be taken daily.
Compliance is especially important for a chronic condition such as hyperlipidaemia, where – as I mentioned earlier – a patient may experience no overt symptoms for years or decades. Keeping a patient’s cholesterol levels consistently low over the course of many years is critical to reducing their risk of cardiovascular events. Yet a patient who does not feel sick may not remember to take their oral medication every day or may be less motivated to adhere to the prescribed treatment regimen, to the detriment of their long-term health.
For example, a 2018 retrospective study in nearly 30,000 hyperlipidaemia patients found that patients who received the correct intensity (level) of statin treatment and complied completely with their treatment had a 40 percent lower risk of cardiovascular events than patients who received low-intensity statin treatment and poor compliance.16 This result illustrates that the right level of treatment and patient compliance are important factors in improving outcomes and saving people’s lives.
As a therapeutic modality, siRNA has many features that make it well-suited for treating the common condition of elevated Lp(a). siRNA’s precise mechanism of gene silencing and liver-specific delivery set it apart from small molecule drugs that target proteins, which can be unpredictable in terms of the tissues they will enter, the metabolites they will form and the side effects they could have, resulting in poor compliance among some patients. Silence believes that its investigational siRNA therapy for elevated Lp(a) has the potential to offer millions of people worldwide a safe and effective option for reducing their risk of cardiovascular events and ultimately save their lives.
Dr Giles Campion is Executive Vice President, Head of Clinical Development and Chief Medical Officer of Silence Therapeutics. Giles is passionate about the company’s vision to transform people’s lives around the world by silencing diseases through precision engineered medicines; specifically, people who have limited or inadequate treatment options.
References
- 1. Centers for Disease Control and Prevention (CDC). Cardiovascular Diseases [internet]. Atlanta, GA: CDC; 2021. Available from: www.cdc.gov/globalhealth/healthprotection/ncd/cardiovascular-diseases.html.
- Kamstrup PR, Benn M, Tybjaerg-Hansen A, Nordestgaard BG. 2008. Extreme lipoprotein(a) levels and risk of myocardial infarction in the general population: the Copenhagen City Heart Study. Circulation. 117 (2): 176-184. doi: 10.1161/CIRCULATIONAHA.107.715698.
- Kamstrup PR, Tybjaerg-Hansen A, Steffensen R, Nordestgaard BG. 2009. Genetically elevated lipoprotein(a) and increased risk of myocardial infarction, JAMA. 301 (22): 2331-2339. doi:10.1001/jama.2009.801.
- Kamstrup PR, Tybjaerg-Hansen A, Nordestgaard BG. 2014. Elevated lipoprotein(a) and risk of aortic valve stenosis in the general population. J Am Coll Cardiol. 63 (5): 470-477. doi:10.1016/j.jacc.2013.09.038.
- Kamstrup PR, Nordestgaard BG. 2016. Elevated lipoprotein(a) levels, LPA risk genotypes, and increased risk of heart failure in the general population. JACC Heart Fail. 4 (1): 78-87. doi:10.1016/j.jchf.2015.08.006.
- Langsted A, Nordestgaard BG, Kamstrup PR. 2019. Elevated lipoprotein(a) and risk of ischemic stroke. J Am Coll Cardiol. 74 (1): 54-66. doi:10.1016/j.jacc.2019.03.524.
- Langsted A, Kamstrup PR, Nordestgaard BG. 2019. High lipoprotein(a) and high risk of mortality. Eur Heart J. 40 (33): 2760-2770. doi:10.1093/eurheartj/ehy902.
- Arsenault BJ, Pelletier W, Kaiser Y, et al. 2020. Association of long-term exposure to elevated lipoprotein(a) levels with parental life span, chronic disease-free survival, and mortality risk: A Mendelian randomization analysis. JAMA Netw Open. 3 (2): e200129. doi:10.1001/jamanetworkopen.2020.0129.
- Rawther T, Tabet F. 2019. Biology, pathophysiology and current therapies that affect lipoprotein (a) levels. J. Mol. Cell Cardiol. 131: 1-11. doi:10.1016/j.yjmcc.2019.04.005.
- Pearson K, Rodriguez F. 2019. Lipoprotein(a) and cardiovascular disease prevention across diverse populations. Cardiol. Ther. 9 (2): 275-292. doi:10.1007/s40119-020-00177-4.
- Gencer B, Kronenberg F, Stroes ES, Mach F. 2017. Lipoprotein(a): the revenant. Eur. Heart J. 38 (2): 1553-1560. doi:10.1093/eurheartj/ehx033.
- Kamstrup PR. 2021. Lipoprotein(a) and cardiovascular disease. Clin. Chem. 67 (1): 154-166. doi:10.1093/clinchem/hvaa247
- Varvel S, McConnell JP, Tsimikas S. 2016. Prevalence of elevated Lp(a) mass levels and patient thresholds in 532,359 patients in the United States. Arterioscler Thromb Vasc Biol. 36 (11): 2239-2245. doi:10.1161/ATVBAHA.116.308011
- Tsimikas S, Stroes ES. 2020. The dedicated “Lp(a) clinic”: A concept whose time has arrived? Atherosclerosis. 300: 1-9. doi:10.1016/j.atherosclerosis.2020.03.003.
- Nordestgaard BG, Chapman MJ, Ray K, et al. 2010. European Atherosclerosis Society Consensus Panel. Lipoprotein(a) as a cardiovascular risk factor: current status. Eur Heart J. 31 (23): 2844-2853. doi:10.1093/eurheartj/ehq386.
- Khunti K, Danese MD, Kutikova L, et al. 2018. Association of a combined measure of adherence and treatment intensity with cardiovascular outcomes in patients with atherosclerosis or other cardiovascular risk factors treated with statins and/or ezetimibe. JAMA Network Open. 1 (8):e185554. doi:10.1001/jamanetworkopen.2018.5554.
