Skip to content

33/163-171 Hawkesbury Road WESTMEAD NSW 2145

Inclisiran and RNAi pathway - new approach to hypercholesterolemia

Inclisiran: From RNA Interference (RNAi) to Cholesterol Management

Last Updated: September 6, 2023 | Dr Reza Moazzeni


Inclisiran is another therapeutic agent that exerts its effect by interfering with the PCSK9 molecule, not by inhibiting its action but by inhibiting its production inside the cytoplasm. Inclisiran uses the body’s naturally occurring machinery called RNA Interference (RNAi) pathway to complete its action. Inclisiran is injected subcutaneously every six months. This has made this medication quite attractive, especially in patients with poor medication adherence.

Inclisiran Mechanism of Action

To fully understand Inclisiran’s mechanism of action, it’s essential to be familiar with the RNAi pathway and to have a basic understanding of genetics. Inclisiran belongs to a rapidly growing category of medical therapeutics known as RNA therapeutics. Within this category, medications that interfere with mRNA are known by the suffix —SIRAN, an anagram for “small interfering (si) RNA.” Examples include Patisiran, a double-stranded RNA that degrades the mRNA responsible for producing the amyloidosis TTR protein, Zilbesiran and Inclisiran. Zilebesiran is an investigational RNA interference therapeutic agent with long-lasting effects that inhibit hepatic angiotensinogen synthesis for the treatment of hypertension.

The mechanism of action for all these RNA interference therapeutics can be divided into two parts. The first is the portion that humans have engineered, and the second leverages the natural RNAi machinery to our benefit. Essentially, by understanding this sophisticated machinery, we can harness its power to make precise and desired changes within the body. However, before discussing the specifics of Inclisiran, let’s discuss the RNAi machinery in further detail.

RNA Interference Pathway (RNAi)

The first hint of RNAi came from plant researchers in the 1980s trying to intensify petunias‘ purple colour. Instead of achieving a deeper purple, some of the transformed plants displayed white petals. Instead, the purple gene was “silenced”, which was unexpected and puzzling.

The groundbreaking work came from Andrew Fire and Craig Mello in the 1990s, who studied the nematode C. elegans. In 1998, they showed that injecting double-stranded RNA (dsRNA) corresponding to a specific gene led to silencing that gene in the worm. Notably, only a tiny amount of dsRNA was required to achieve this effect, suggesting that the dsRNA was somehow being amplified or catalysing a more massive response. Fire and Mello were awarded the Nobel Prize in Physiology or Medicine in 2006 for this discovery.

RNA inhibition (RNAi) pathway discovery by Craig Mello and Andrew Fire
Andrew Fire and Craig Mello

Following this discovery, many researchers studied the RNAi pathway to understand the mechanism better. It became clear that, upon entering the cell, dsRNA was processed by an enzyme called Dicer into small interfering RNAs (siRNAs). These siRNAs would then guide the RNA-induced silencing complex (RISC) to its target mRNA, leading to the degradation of the target and preventing its translation into protein.

By discovering this natural pathway, we disclosed a vast domain of opportunities. The steps seem straightforward: choose a protein, decode its mRNA, and craft a complementary dsRNA in the lab. To ensure it reaches the right organ, like the liver, we take a unique receptor specific to that organ and then attach a tailored ligand to our engineered dsRNA. Now, by engineering different dsRNAs, we have potential game-changers: Inclisiran targets PCSK9 mRNA to manage hypercholesterolemia, Zilebesiran focuses on angiotensinogen mRNA to treat hypertension, and Patisiran targets TTR protein mRNA, putting the brakes on TTR protein production in Amyloidosis.

Here’s a breakdown of the RNAi pathway and its components (Fig-1):

1. Initiation of RNAi: Double-Stranded RNA (dsRNA) Introduction

The RNAi machinery is initiated when cells encounter dsRNA. This dsRNA can originate from various sources: it could be from viruses, transposable elements —Jumping genes—or it can be introduced artificially for research or therapeutic purposes such as Inclisiran. dsRNA ignites this inhibitory pathway, regardless of the genetic code it carries, which makes the system capable of inhibiting a wide range of messenger RNAs.

2. Dicer: The Molecular Scissors

In the next step, an enzyme called Dicer cleaves the long dsRNA molecules into smaller fragments known as small interfering RNAs (siRNAs). These siRNAs are about 20-25 nucleotides in length.

3. RISC Assembly: The Executioner Complex

The siRNA is then incorporated into a complex of proteins called RNA-induced silencing complex (RISC). However, before its full activation, the siRNA double-strand is unwound, and the sense strand is removed. The remaining antisense strand, known as the “guide” strand, stays bound to RISC.

The key component of RISC is a protein from the Argonaute (Ago) family that keeps the complex together. RISC is a tool that cells use to find and destroy specific messages (mRNA) that are used to make proteins. One siRNA-RISC unit can target multiple mRNAs. Additionally, specific modifications to Inclisiran’s dsRNA boost its stability and longevity, contributing to the drug’s ling-acting effects.

4. Target mRNA Cleavage

Once the RISC is activated, it goes on the mission to find the matching mRNA to the siRNA that it carries. After finding and binding to the target mRNA, the Argonaute cleaves the target mRNA, leading to its degradation and preventing its translation into protein. Inclisiran uniquely targets the PCSK9 mRNA, thereby inhibiting the synthesis of the PCSK9 protein. For a better understanding of the mechanism of action of PCSK9 and how PCSK-9 inhibition results in reduced LDL-C levels, refer to my previous post.


Fig-1: RNAi Pathway (Click to enlarge)

Inclisiran’s action to inhibit PCSK9 production begins with a subcutaneous injection of a double-stranded RNA (dsRNA) engineered complementary to the PCSK9 mRNA. This dsRNA is bound to N-acetylgalactosamine (GalNac), which plays a pivotal role by acting as a high-affinity ligand for the liver-specific asialoglycoprotein receptor (ASGRP) that ensures the liver swiftly takes up the drug. Clinical trials have shown the efficacy of this targeting, with no free Inclisiran detected in the plasma after 48 hours post-administration. Once bound to the ASGRP receptor, the next phase of action kicks off: the dsRNA is internalized into the cell through endocytosis. Within the cellular endosome, the constituents are sorted; the receptor is primed for recycling back to the cell surface, and the dsRNA is prepped for the next step.
An enzyme named Dicer takes charge at this juncture, cleaving the dsRNA into smaller RNA fragments known as small interfering RNA (siRNA). The siRNA pairs with a protein complex to form the RNA-induced silencing complex (RISC). This complex undergoes a brief maturation process, during which the two strands of the siRNA are separated. The “sense” strand is marked for degradation, leaving the “antisense” strand ready for its guiding role. With the antisense strand leading the way, RISC seeks out its target: the PCSK9 mRNA. Upon locating and binding to it, a component of the RISC, the Ago protein, cleaves the mRNA. This critical step ensures the mRNA is incapacitated, preventing it from being translated into the PCSK9 protein molecule.

RNA Interference role in our body

The RNAi system serves several crucial roles in our bodies, among which the following two natural roles stand out:

  • Defending Against RNA Viruses: When an RNA virus infects us — examples include common cold, influenza, SARS, MERS, Covid-19, Dengue Virus, hepatitis C, hepatitis E, West Nile fever, Ebola virus disease, rabies, polio, mumps, and measles — the virus introduces its genome, often in the form of double-stranded RNA. RNA interference degrades the viral RNA, halting the production of new virus particles.
  • Regulating Gene Expression: Our bodies sometimes need to dial down the production of specific proteins. To achieve this, our DNA dispatches microRNAs. Once in the cytoplasm, these microRNAs can form dsRNA structures that target and neutralize specific previously made mRNAs, preventing their translation into proteins.

Additionally, scientists can design specific dsRNA molecules in research and therapeutic settings. These tailor-made dsRNAs are employed to engage the RNAi machinery and degrade mRNAs to particular genes, either to study their function or as potential therapeutic interventions. (Fig-2)

Fig-2: RNAi Pathway roles in our cells (Click to enlarge)

The RNAi system plays two pivotal natural roles: defending against viruses and regulating gene expression. Exploiting this system to our benefit, we’ve engineered RNA templates to precisely silence specific genes and their resultant proteins.

Why a double-strand RNA has been chosen for Inclisiran rather than single-stand?

In the groundbreaking experiments conducted by Andrew Fire and Craig C. Mello, single-stranded RNA didn’t produce the desired gene-silencing effect in nematode worms. However, when they introduced dsRNA corresponding to a specific gene, they witnessed a strong interference effect, resulting in a phenotype, in this case, worm twitching.

Using dsRNA emulates the natural RNA interference process, often triggered by dsRNAs, thus tapping into our cells’ innate recognition system. Dicer cleaves dsRNA —not single strand— into small interfering RNAs (siRNAs). Using dsRNA ensures the endogenous Dicer processing produces the desired siRNA, priming it for efficient RISC loading. dsRNAs are more stable than single-stranded RNAs, offering protection against degradation. Their structure is also more readily recognized by cells, aiding in efficient cellular uptake. While inside the cytoplasm, with two strands —sense and antisense— there’s a higher chance the antisense strand will remain intact for RISC loading, even if there’s partial degradation, increasing the longevity of the molecule with long-lasting effects. In essence, dsRNA offers a more efficient, stable, and natural method of leveraging the RNAi pathway, making it a preferred choice for therapies like Inclisiran.

Why are statins still recommended for patients who are already on PCSK-9 inhibitors like Inclisiran and Evolocumab?

Statins and PCSK-9 inhibitors function through distinct mechanisms to reduce LDL-C, and their combined use can have synergistic benefits. Statins primarily inhibit the enzyme HMG-CoA reductase in the liver. This inhibition leads to an upregulation of LDL receptors, which aids in the increased clearance of LDL. However, as a feedback mechanism, the increased LDL receptor expression from statin use also results in an upregulation of PCSK9 production. Elevated PCSK9 can lead to faster degradation of LDL receptors, potentially limiting statins’ full LDL-C lowering potential.

Inclisiran reduces PCSK9 production and, consequently, LDL receptor degradation. This action preserves and even amplifies the number of available receptors to clear LDL. Using them in tandem allows for a profound reduction in LDL-C levels. This combination is particularly beneficial for patients with very high baseline LDL-C or those at elevated cardiovascular risk. (Fig-3)

Fig-3: The different and complementary mechanisms of action of statins and Inclisiran make them an excellent combination therapy for lowering LDL-C levels. (Click to enlarge)

Inclisiran dose and effect on Lipids and Apolipoproteins

Inclisiran is administered subcutaneously in two initial doses of 300 mg on days 1 and 90, followed by injections every six months. This regimen achieves an LDL-C reduction of roughly 50%, complementing the effects of other lipid-lowering drugs like statins. Moreover, other significant reductions were observed: 40% in ApoB, 42% in Non-HDL-C, 13% in triglycerides, and 20% in Lp(a) or Lipoprotein(a).

Reduction from baseline (%)
LDL-C 50
Apo-B 40
Non-HDL-C 42
Triglycerides 13
Lp(a) 20

Fig-4: Percentage change of LDL-C over time with two doses of Inclisiran (Click to enlarge)

In 300 mg dose group, three months after the first injection, there was a 45% reduction in LDL-C and after six months, about 54%. At day 360, there was still a 32% reduction in LDL-C. From Ray KK, Stoekenbroek RM, Kallend D, et al. Effect of 1 or 2 doses of inclisiran on low-density lipoprotein cholesterol levels: one-year follow-up of the ORION-1 randomized clinical trial. JAMA Cardiol. 2019;4(11):1067–1075

Inclisiran Safety and Side Effects

Inclisiran has been studied in multiple clinical trials to determine its safety and efficacy. Generally, it has shown a favourable safety profile, but like all medications, there are potential side effects.

  • Injection-site reactions: Patients on Inclisiran had slightly more injection-site reactions at 5.0%, compared to 0.7% with the placebo. These reactions typically included pain, erythema (redness), rash, pruritus (itchiness), and hypersensitivity. However, these reactions were usually mild to moderate in severity and were nonpersistent. For those who continued Inclisiran after an injection-site reaction, the severity remained the same, with only 0.3% showing increased severity and 0.2% showing a decrease.
  • Respiratory issues: In pooled analyses from certain clinical trials, there was a slightly higher incidence of bronchitis in the Inclisiran group compared to the placebo. However, rates of other respiratory tract infections did not show significant differences between the Inclisiran and placebo groups.
  • General safety: Overall, in several studies, the occurrence of adverse events was comparable between Inclisiran and placebo groups, suggesting that the drug doesn’t significantly increase the risk of adverse events.
  • Long-term safety: While initial studies show a good safety profile for Inclisiran, like with all the new medications, ongoing post-marketing surveillance and long-term studies will provide more information about any potential rare side effects and the drug’s safety over time.

Inclisiran Pivotal Trials

Based on data from inclisiran’s clinical development program— ORION — Inclisiran was approved, under the name Leqvio, as a cholesterol-lowering agent in December 2020 in Europe and in December 2021 in the USA. It is indicated for use in adults with primary hypercholesterolemia (heterozygous familial or nonfamilial) or mixed dyslipidemia if LDL-C goals are not achieved through diet and maximally tolerated doses of statins or with other lipid-lowering agents, such as Bempedoic acid, if statins are not tolerated or contraindicated. Similar to other lipid-lowering drugs, Inclisiran has been approved for lipid-lowering. However, trials with clinical event endpoints are still ongoing. The results of the cardiovascular outcome trial ORION-4, which includes over 15,000 patients, are expected in 2024.

Trial Patients End Points
ORION-1 501 patients with established ASCVD or FH percentage change in LDL-C from baseline to day 180
ORION-9 482 patients, Genetically confirmed HeFH or positive clinical criteria (DLCN >8 points, or untreated LDL-C >4.9 mmol/L + family history of FH, or elevated cholesterol, or early heart disease) and LDL-C ≥2.6 mmol/L despite maximally tolerated statins ± ezetimibe 48% LDL-C reduction from baseline to day 510
ORION-10 1561 patients with established ASCVD and LDL-C ≥1.8 mmol/L despite maximally tolerated statins with or without ezetimibe 52.3% LDL-C reduction from baseline to day 510
ORION-11 1617 patients with established ASCVD or ASCVD risk equivalents (T2D, FH, 10-year risk of ASCVD risk score ≥20%) and LDL-C ≥1.8 mmol/L despite maximally tolerated statins with or without ezetimibe 49.9% LDL-C reduction from baseline to day 510
ORION-4 15,000 patients with Established ASCVD MACE: CHD death, myocardial infarction, fatal or nonfatal ischemic stroke, or urgent coronary revascularization procedure
ORION-5 56 patients with Genetically confirmed HoFH or untreated LDL-C >13 mmol/L + xanthoma before 10 years of age or HeFH in both parents and LDL-C ≥3.4 mmol/L Percentage change in LDL-C from baseline to day 150

Inclisiran Long-lasting Effects and Improved Patients Adherence

Treating chronic, often asymptomatic conditions, such as hypercholesterolemia or hypertension, presents a unique challenge for both clinicians and patients. Enhanced adherence often necessitates a combination of comprehensive patient education, fixed-dose regimens, pill reminders, and other innovative strategies.

However, non-adherence remains particularly high among patients prescribed small-molecule tablets like statins, ezetimibe, or bempedoic acid. Even with the two-weekly or monthly dosing of PCSK9 monoclonal antibodies, data indicates about 30% of patients discontinue therapy after just 6 months, and this number jumps to 50% after 3 years.

Inclisirans extended duration of action offers a significant advantage in patient adherence due to its infrequent dosing schedule (Fig-5). Moreover, since inclisiran is administered by healthcare professionals, there’s an added layer of surveillance, ensuring the drug is delivered as intended. This enhanced adherence can significantly decrease long-term exposure to LDL-C, consequently reducing the risk of ASCVD. It’s worth noting that lapses in adherence to lipid-lowering therapies are estimated to cause 12,000 avoidable cases of atherosclerotic cardiovascular disease for every 500,000 patients annually.

Fig-5: Inclisiran infrequent dosing and improved adherence (Click to enlarge)

The medication burden increases non-adherence, which impairs LDL-C reduction over time. This picture shows annual medication burdens for lipid-lowering regimens. The highest medication burden is with statins. New therapeutic agents are associated with lower administration frequency. Near-perfect adherence is feasible with infrequent dosing regimens (siRNAs, vaccines) administered by medical professionals and permanent interventions such as gene editing. The impact of adherence on average LDL-C reduction is shown in yellow. Assuming an annual average LDL-C reduction of 39 mg/dL with perfect adherence (siRNAs, vaccine, gene editing), mAbs with imperfect adherence will maintain an annual average LDL-C reduction of 31 mg/dL, and statins an average LDL-C reduction of 23 mg/dL. From Brandts J, Ray KK. Low-density lipoprotein cholesterol-lowering strategies and population health: time to move to a cumulative exposure model. Circulation. 2020;141(11):873–876.


In summary, inclisiran, utilizing the RNAi pathway, presents a promising advancement in the management of hypercholesterolemia. Its biannual dosing regimen offers the potential for increased patient adherence, coupled with a favourable safety profile. As evidenced by inclisiran, RNA-based therapeutics herald a transformative potential in the forthcoming chapters of medical science.

References and further reading

0 0 votes
Article Rating
Notify of

Inline Feedbacks
View all comments

What is Atrial Fibrillation (AF or AFib)

Recent posts

CT Coronary Angiogram vs CT Calcium Scoring

The components of a healthy lifestyle

Is my high cholesterol genetic?

Would love your thoughts, please comment.x