siRNAs: the future of cholesterol management?

By Allis Lai

On September 1, 2021, NICE approved inclisiran, a new siRNA drug for patients with primary hypercholesterolaemia or mixed dyslipidaemia.¹ This is a big step forward for RNA therapeutics, a field that has been rapidly gaining traction in the past two decades. Small interfering RNA, known in short as siRNA, is a short double stranded RNA molecule that leads to targeted gene suppression via RNA interference (RNAi). Upon entering the cytoplasm, endoribonuclease Dicer cleaves the siRNA molecule, producing mature siRNA of 21-23 bases in length. This mature siRNA is bound to the RNA-induced silencing complex (RISC) consisting of integral proteins. The siRNA dissociates into the sense and antisense strand. The former is cleaved from the RISC, whilst the latter acts as a guide to direct the RISC to the target mRNA sequence. Binding of the antisense strand to the target gene triggers its cleavage, thereby achieving gene suppression.² 

Despite knowledge of the RNAi mechanism for almost two decades, siRNA therapeutics only became approved in the recent few years. The main challenges to development of safe siRNA drugs was achieving site specific delivery: at a systemic level, siRNA molecules are rapidly cleared by the kidneys and broken down by nucleases in plasma, tissues and cytoplasm. After that, they must pass through the adherence and tight junctions of the capillary endothelium and be taken up across cellular membranes. Furthermore, siRNA molecules run the risk of activating the immunogenic response if they are recognised as viral molecules by immune mediators. To overcome these barriers, scientists have trialed and made many modifications to siRNA molecules, one of which being replacing the phosphodiester backbone with phosphorothioate. This increases resistance to nuclease activity and increases hydrophibicity, which promotes siRNA binding to carrier plasma proteins, overall leading to slower breakdown. These developments in siRNA drugs gives scientists the opportunity to selectively act on previously “undruggable” targets by acting at the gene level rather than the protein level.² 

Until recently, most siRNA drugs focused on treating rare “orphan diseases”. ²The approval of inclisiran in treating hypercholesterolaemia therefore marks a milestone in expanding RNAi application in modern healthcare. Hypercholesterolaemia, an increase in serum levels of low-density lipoprotein cholesterol (LDL-C) is a prevalent disorder and leads to increased risk of cardiovascular disease. Primary hypercholesterolaemia (FH) is usually associated with genetic causes, which is known as familial hypercholesterolaemia. Autosomal dominant familial hypercholesterolemia affects 1 in every 250 individuals, and the main genes involved are the apolipoprotein B (APOB), PCKS9, and LDLR (LDL receptor) genes. ³ About 90% of FH is caused by mutations in LDLR, 5% by mutations in APOB, and 1-2% due to mutations in PCSK9.⁴ Inclisiran targets the PCKS9 gene to achieve its therapeutic effect. 

PCSK9 gene encodes the proprotein convertase subtilisin/kexin type 9 protein, a protein which binds to LDL to regulate the number of LDLR present on hepatocytes. In normal lipid regulation, LDL particles bind to LDLR on hepatocytes via the apoB molecule. The LDL-C complex is internalised and degraded in lysosomes, whilst LDLR is returned to the membrane and recycled.⁴ PCSK9 disrupts this process via preventing LDLR release, increasing its degrdation in endosomes and lysosomes, thereby reducing LDL-C uptake in the liver. PCSK9 has also been suggested to have a role in accelerating atherosclerosis via other mechanisms involving inflammation and hypertension.³ 

The current treatment standard for primary hypercholesterolaemia is a combination of dietary changes, statins and other cholesterol lowering drugs.¹ Statins are a class of drugs that inhibit HMG-CoA reductase, an enzyme involved in cholesterol synthesis in the liver to decrease serum LDL-C levels⁴. Monoclonal antibody PCSK9 inhibitors such as alirocumab and evolocumab may also be used. These are IgG antibodies self-administered subcutaneously every two or four weeks which bind to PCSK9, preventing its binding to LDLR so that LDLR can be recycled. Though typically well tolerated, some adverse effects include injection site reactions like itching, redness and swelling.⁵ Being an antibody-based approach, it’s main limitation is that it can only capture circulating PCSK9 to prevent its binding to LDLR, but cannot influence the intracellular portion of protein inside the cell.⁶ Though the clinical value of intracellular PCSK9 remains unknown, it has been suggested that though entry of PCSK9 into cells is dependent on LDLR, internalised PCSK9 remains intact intracellular for several hours and there may be a recycling system fo rintracellular PCSK9 to return to surface and act on new LDLR.⁷ Inclisiran, being an RNA based approach, is able to affect both extracellular and intracellular PCSK9 levels, leading to long-lasting reduction of serum cholesterol concentration.⁶ 

Inclisiran is a siRNA consisting of a 23-base guide strand and 21-base passenger strand.² After it is incorporated into the hepatocyte, the guide strand binds to the RISC and undergoes hybridization with the complementary mRNA for PCSK9, inducing its degradation. The RISC remains active after mRNA degradation, meaning a single siRNA molecule can suppress expression of multiple mRNA molecules. In studies with non-human primates, it showed promising results, leading to reduction of PCSK9 levels by over 80% and reduction in serum LDL-C levels by 60%.² PCSK9 inhibition is not expected to have significant consequences, as it was found that people with loss of function variants of the PCSK9 variants remained healthy despite very low LDL-C levels.⁷ In clinical trials, it was well tolerated as well with an adverse effect rate comparable to that of the placebo group ² and those associated with anti-PCSK9 monoclonal antibodies.⁶  

Furthermore, inclisiran offers an additional benefit over monoclonal antibodies: it does not affect PCSK9 production in atheromas. Both plaque macrophages and smooth muscle cells in atheromas secrete and are responsive to circulating PCSK9, which regulates LDLR expression in atheroma cells. Contrary to the liver, uptake of LDL-C in atheromas are not desirable as it leads to increased cholesterol accumulation. Monoclonal antibodies block PCSK9 action in the atheroma. SiRNA intervention, however, does not affect PCSK9 production in the atheroma due to specific uptake in the liver, although it does decrease circulating PCSK9 levels.⁷  

Inclisiran appears to be a promising cholesterol-lowering drug. Economically, it is predicted to do well against its competitors (namely alirocumab and evolocumab) as it is cheaper to synthesise and has a less frequent dosing period of twice a year, making it more appealing to patients.² Despite not having sufficient long-term evidence supporting its efficacy in decreasing CVD risk, it finds its value as a potential treatment for patients who did not respond well to existing alternatives. 

References:  

1. NICE approves ground-breaking cholesterol-lowering drug inclisiran | News and features | News [Internet]. NICE. 2021 [cited 2022 Mar 17]. Available from: https://www.nice.org.uk/news/article/nice-approves-ground-breaking-cholesterol-lowering-drug-inclisiran 
2. Zhang MM, Bahal R, Rasmussen TP, Manautou JE, Zhong X. The growth of siRNA-based therapeutics: Updated clinical studies. Biochemical Pharmacology. 2021 Jan;114432.  
3. Guo Q, Feng X, Zhou Y. PCSK9 Variants in Familial Hypercholesterolemia: A Comprehensive Synopsis. Frontiers in Genetics. 2020 Sep 23;11. 
4. Pang J, Chan DC, Watts GF. The Knowns and Unknowns of Contemporary Statin Therapy for Familial Hypercholesterolemia. Current Atherosclerosis Reports. 2020 Sep 1;22(11). 
5. Kaddoura R, Orabi B, Salam AM. Efficacy and safety of PCSK9 monoclonal antibodies: an evidence-based review and update. Journal of Drug Assessment [Internet]. 2020 Aug 11 [cited 2022 Mar 17];9(1):129–44. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470150/ 
6. Dyrbuś K, Gąsior M, Penson P, Ray KK, Banach M. Inclisiran—New hope in the management of lipid disorders? Journal of Clinical Lipidology. 2020 Jan;14(1):16–27. 
7. Rosenson RS, Hegele RA, Fazio S, Cannon CP. The Evolving Future of PCSK9 Inhibitors. Journal of the American College of Cardiology [Internet]. 2018 Jul [cited 2022 Mar 17];72(3):314–29. Available from: http://www.onlinejacc.org/content/72/3/314?_ga=2.7004854.548350756.1572404618-2034167674.1564383832