Non-coding RNA therapeutics in cardiovascular diseases and risk factors: Systematic review

At present, RNA-based therapy which includes therapies using non-coding RNAs (ncRNAs), antisense oligonucleotides (ASOs), and aptamers are gaining widespread attention as possible ways to target genes in various cardiovascular diseases (CVDs), thereby serving as a promising therapeutic approach for CVDs and risk factors management. However, data are primarily in an early stage. A systematic review was carried out using literature from several databases (Pubmed, Cochrane, Scopus, and DOAJR) following the PRISMA guidelines. Of the 64 articles reviewed, 39 papers were included in this review with three main types of RNAs: aptamers, antisense oligonucleotides (ASOs), and small-interfering RNA (siRNA). All studies were human clinical trials. RNA-based therapies were demonstrated to be efficacious in treating various CVDs and controlling cardiovascular risk factors. They are generally safe and well-tolerated. However, data are still in the early stage and warrant further investigation.


Introduction
Despite the current advance in therapeutic strategies, cardiovascular diseases (CVDs) are still a leading cause of mortality worldwide, and CV risk factors such as hyperlipidemia, diabetes, and hypertension further contribute to the progression of CVDs [1]. RNA-based therapeutics have emerged as a vastly growing field that holds promising value in diagnosing and managing diverse health conditions, including CVDs and management of CV risk factors. Different types of RNA have been known to play a crucial role in regulating gene expression that is responsible for health outcomes in many CVDs [2,3]. Exogenous RNA agents offer varying mechanisms of action, including gene silencing, mRNA editing, or replacement, which further broaden the possibility of addressing different pathophysiological pathways in varying diseases. Thus, this novel therapy may aid in the treatment and prevention of CVDs along with managing its risk factors [4].
Several strategies have been developed to target gene expression including the use of antisense oligonucleotides (ASO), aptamers, smallinterfering RNA (siRNA), microRNA (miRNA), and messenger RNA (mRNA) [2,3]. siRNA and miRNA target specific endogenous mRNA to inhibit the subsequent protein translation. In contrast, ASOs bind to mRNA to block its function [5]. A newly-invented agent, aptamers, also binds to specific target molecules through a repetitive process termed systemic evolutions of ligands by exponential enrichment (SELEX) [6]. Meanwhile, mRNA therapy utilizes modified mRNA to directly encode functional proteins to cure certain conditions. This approach demonstrated excellent therapeutic results in several different CVDs, such as atrial fibrillation and coronary artery diseases [7]. Furthermore, the use of RNA-based therapy also manages risk factors in CVD, such as hyperlipidemia and diabetes mellitus [8,9].
Despite all this exciting progress, many caveats still need to be addressed before widely implementing RNA-based therapy in clinical settings. This study aims to evaluate and summarize current advances in the field of RNA-based therapy for the treatment of CVDs and provide a comprehensive overview regarding the use of non-coding RNA therapeutics in managing CVDs and their risk factors. the study. We reported the mean difference between treatment and control groups on primary or related outcomes.
Then, using the Cochrane risk-of-bias tool for randomized trials (RoB 2) for RCTs and the Risk Of Bias In Non-Randomized Studies -of Interventions (ROBINS-I) for non-randomized clinical trials, two reviewers independently assessed the risk of bias. RoB 2 is classified into five main domains, namely assessment of bias arising from; (1) The randomization process, (2) Deviations from intended interventions, (3) Missing the outcome data, (4) Measurement of the outcome, and (5) Selective reporting. Meanwhile, ROBINS-1 divides into seven domains assessing bias from confounding factors, selection of participants, intervention classification, missing data, measurement of outcomes, selective reporting, and other sources of bias. The assessment will be summarized in the form of a Risk-of-Bias visualization tool (Robvis).

Antisense oligonucleotides (ASO)
Antisense oligonucleotides (ASO) is a synthetic agent made of short single-stranded DNA-based oligonucleotides binding to target mRNA to inhibit protein translation via Watson-Crick base-pairing and typically has 20 base pairs [10]. ASO targets various classes of nucleic acids, such as mRNA, pre-mRNA, and non-coding RNA. It allows the inhibition of protein production by forming a duplex with the target mRNA and stimulating RNAase H that degrades the target mRNA. RNA/ASO duplex can alternatively block pre-mRNA processing and mRNA translation [5] (Fig. 2). ASOs are subject to modifications, which may improve the pharmacodynamic and pharmacokinetic properties. It has been studied for both neurodegenerative diseases and cardiovascular diseases (CVDs) [2]. Table 1 illustrates the use of ASO as therapy in studies of CVDs and risk factors, drug monitoring, and outcomes, including death, MACE, and allergic reactions. The most studied CVDs were hypercholesterolemia (55%,11/20 studies), transthyretin amyloidosis (20%, 4/20 studies), atherosclerotic cardiovascular disease (ASCVD), and atrial fibrillation. The most studied ASOs were mipomersen (20%, 5/20 studies) and inotersen (15%, 3/20 studies).

Hypercholesterolemia
The most studied CVD risk factor is lipid disorders which cover a wide range of diseases (e.g. hypertriglyceridemia, hypercholesterolemia, elevated lipoprotein(a), etc.). Plasma triglycerides contain triglyceride-rich lipoproteins and lipoprotein particles. The elevation of plasma triglyceride-rich lipoproteins and the remnants is correlated to ASCVD. Its elevation also increases VLDL, chylomicron, and LDL-C, activating cholesteryl ester transfer protein. The liver produces very low-density lipoprotein particles. They contain apolipo-protein B-100, C-I, C-II, C-III, and E. LDL is a lipoprotein most abundant with apoB-100, a known modifiable risk factor of ASCVD targeted for therapies. Part of the LDL is lipoprotein (a), an LDL particle with apolipoprotein (a) attached to apo B-100 via a single disulfide bond [11,12]. ASOs target different checkpoints or proteins in lipid metabolism, such as apoCIII, apo(a), apo(b), and ANGPTL3 [13].
The liver produces the amino acid glycoprotein apoCIII, which is connected to apoB and HDL. ApoCIII affects serum triglyceride levels and obstructs the clearance of triglyceride-rich lipoproteins via both LPL-dependent and -independent pathways. Hence, when projected on LDL and HDL, the elevated level of apoC-III predicts skyrocketing CVD risk. Volanesorsen is a second-generation ASO directed to mRNA of apoCIII that has been proven to reduce plasma apoCIII and triglyceride levels by ~70% in animal and human models. Phase II clinical research conducted by Yang et al. [14] later revealed that hypertriglyceridemic individuals had a significant reduction in apoC-III on apoB-100, apoC-III-Lp(a), and apo(a)-I lipoproteins (reduction of treatment arm vs placebo; 82.3 ± 11.7%, 81.3 ± 15.7%, and 80.8 ± 13.6% respectively, p < 0.001 for all). This study implies the potency of Volanesorsen to reduce triglycerides and apoC-III-mediated cardiovascular risk.
Another important particle in lipid metabolism is apoB, an essential structural protein of all atherogenic lipoproteins (i.e. LDL-C, IDL-C, VLDL-C, Lp(a)). Mipomersen, an ASO that inhibits the synthesis and secretion of apoB, has been widely studied for the past decade [15,16]. Mipomersen binds to the apoB mRNA in the liver, causing mRNA degradation and, as a result, the cessation of apoB protein translation [17]. Several phase III trials have been conducted in different populations. Thomas et al. [8] tested mipomersen in a population of non-familial hypercholesterolemia with high CVD risk due to coronary heart disease or type 2 diabetes mellitus (T2DM), which resulted in a significant reduction of LDL-C (mean difference treatment vs placebo, − 38 95% CI -49.3824 to − 26.6176, p < 0.001). The apoB and Lp(a) also decreased in number. However, 96% (97/101) of subjects experienced mild adverse events related to treatment (e.g. injection site reaction, flu-like symptoms to hepatic steatosis) [13]. Santos et al. [18] conducted four mipomersen phase III trials in people with severe hypercholesterolemia, homozygous familial hypercholesterolemia (FH), heterozygous FH with concurrent coronary artery disease (CAD), and hypercholesterolemia with a high risk for CAD. All his studies reported that mipomersen significantly reduced all LDL particles and Lp(a) concentrations, with an adverse event of injection site reactions. Previously, three phase II studies reported a similar trend of decreased concentration of apoC-III, LDL-C, and apoB [15,16,19]. However, the rate of side effects was high, ranging from mild to moderate adverse events (i.e. site injection reactions, flu-like symptoms, and elevated liver enzymes).

Atrial fibrillation
Atrial fibrillation is a common cardiac arrhythmia that involves complex pathophysiology, of which the exact etiology remains unclear. Inflammation is believed to play a significant role in the development of AF. Thus, the reduction of any inflammatory markers has been found to suppress AF development [7]]. A second-generation 2′-O-(2-methoxyethyl) chimeric antisense oligonucleotide (ASO) called ISIS-CRPRx       (ISIS-329993) inhibits CRP in order to function. CRP is an acute-phase protein that was proposed to be able to promote atrial fibrosis, oxidative stress, and electrical remodeling. Tanaka et al.'s cohort study found a strong correlation between raised hs-CRP levels and an increased risk of atrial fibrillation in the Japanese population [20]. Similar cohort research conducted by Lee Y et al.'s discovered that chronically increased CRP levels independently predicted the development of AF [21]. In a phase II RCT by Sugihara et al. [22] this medication has been utilized for AF patients with dual chamber permanent pacemakers (PPM) and was generally well tolerated with no significant side events in the study population. However, the study yielded no significant result in reducing AF burden (treatment arm vs.

The use of ASO in venous thromboembolism
Factor XI Antisense Oligonucleotide (FXI-ASO) demonstrated a positive outcome in one phase II RCT by Büller et al. [24] in the prevention of venous thrombosis. FXI-ASO (ISIS 416858) is a second-generation ASO, which works by inhibiting Factor XI production, thereby hampering the blood coagulation process. A total of 281 subjects who underwent total knee arthroplasty were randomized to receive either 200 mg FXI-ASO, 300 mg FXI-ASO, or 40 mg Enoxaparin as a comparison. Subjects in the treatment arms received either 200 or 300 mg of FXI-ASO, while subjects in the comparison arm were given 40 mg of Enoxaparin. The result showed that 300 mg of FXI-ASO had a significantly lower incidence of venous thromboembolism compared to the Enoxaparin group (FXI-ASO vs. Enoxaparin; n = 3 [4%], 95% CI 1 to 12 vs. n = 21 [30%], 95% CI 20 to 43, p = <0.001) with a risk difference of − 26 (upper limit of 95% CI -16). On the other hand, 200 mg of FXI-ASO did not show any superiority compared to Enoxaparin (p = 0.59). Serious adverse events occurred in four subjects and two subjects needed to discontinue the use permanently due to worsening arterial hypertension and bleeding from the surgical site [24].

The use of ASO in ATTR
Amyloidosis is a disease caused by a buildup of an abnormal protein named amyloid in body organs [25]. Transthyretin protein, or TTR, misfolds, reshapes, and forms fibrous clumps that are deposited in many parts of the body, resulting in ATTR amyloidosis, a serious systemic illness [26]. Most patients manifest signs and symptoms of nerves and heart, although other organs may be involved. ATTR can be inherited in an autosomal dominant fashion. According to research by Tanskanen et al., 25% of myocardial samples from individuals older than 85 years old showed ATTR amyloid deposits [27]. Restrictive cardiomyopathy is caused by myocardial ATTR amyloid deposition and is characterized by the thickness of the biventricular wall, stiffness of the myocardium, and the emergence of systolic and diastolic dysfunction. Recently, pharmacological therapy to improve outcomes and prolong survival has emerged. Reducing TTR synthesis and interrupting the appropriate mRNA by preventing dissociation into amyloidogenic monomers or cleavage into amyloidogenic fragments are two therapeutic methods that are intended to lessen continuing ATTR amyloid fibrillogenesis [28,29]. One of which is ASOs targeting mRNA of mutant transthyretin genes to suppress the hepatic production of TTR called Inotersen or IONIS-TTRRx [25]. An identical sequence and similar design of ASO targeting TTR mRNA is AKCEA-TTR-LRx, whose particles are conjugated to a GalNAC3 ligand to accelerate hepatocyte uptake [30]. When ASO binds to complementary mRNA, it causes the mRNA to degrade in several different ways, including sterically inhibiting ribosome attachment and encouraging endogenous ribonuclease H1-mediated breakdown. Without a transfection reagent, ASO can penetrate cells [28]. Phase III trials of inotersen in ATTR patients with polyneuropathy reported an improved quality of life with several adverse events ranging from glomerulonephritis to thrombocytopenia and death. Out of 5 deaths, one was related to grade 4 thrombocytopenia, while the others were unrelated to therapy [30]. Phase II trials of inotersen yielded a similar trend in which disease progression is limited and cardiac amyloid burden reduced. This trial did not record adverse renal effects; however, one sudden cardiac death post-emergency cholecystectomy was reported [31]. The phase I trial reported several adverse events, such as decreased renal function (one patient) and declined platelet level, although no grade 4 thrombocytopenia. The cardiac parameters measured by left ventricular function and cardiac MRI were found to be constant or increased [32]. The effect of lowering TTR on cardiac amyloidosis warrants further investigations. No serious adverse events were recorded during the AKCEA-TTR-LRx phase I trial, demonstrating the drug's safety; additionally, there was a significant drop in TTR levels. Further study is required to evaluate the efficacy of AKCEA-TTR-LRx [33].

Aptamer
Globally, cardiovascular disease has a high mortality rate, which requires the development of novel diagnostic and therapeutic methods. Aptamers are single-stranded oligonucleotides that can compete with antibodies in cardiac applications as they uniquely recognize and bind targets by forming unique structures in vivo [34]. Major cardiovascular events are frequently caused by thrombus development. Therefore, the alternative treatment option might involve using RNA aptamers as a reversible FIXa inhibitor [6]. A novel RNA-aptamer-based FIXa inhibitor is named pegnivacogin (RB006) [35]. An ASO, anivamersen (RB007), improves hemostasis by blocking pegnivacogin from binding to Factor IXa [35] (Fig. 3).
Antiplatelet therapy is used to treat ischemic disorders, with the main emphasis being on the activation and aggregation of platelets rather than the activity of von Willebrand Factor (vWF) [6,36]. However, since vWF is an important factor in atherogenesis and artery circulation, it may be a target for cardiovascular disease intervention. vWF has a unique role in thrombus generation, and as an aptamer targeting molecule, has shown early success in antithrombotic treatment [6]. The GPIb-vWF pathway has been blocked by anti-vWF aptamers [37]. Without causing significant bleeding, ischemia events, or other severe negative effects, factor IXa was successfully inhibited with an RNA aptamer, and its coagulant activity was actively restored with an antidote [35].

The use of aptamer in healthy volunteers
Staudacher et al.'s study [35] found that the new RNA-aptamer-based FIXa inhibitor pegnivacogin reduced platelet reactivity in blood samples from healthy volunteers. In this work, platelet aggregation calculations were used after ex-vivo incubation with pegnivacogin [35]. According to Arzamendi et al. [38], platelet adhesion is reduced by ARC1779, an aptamer to the VWF A1 domain, in healthy subjects without having a meaningful impact on P-selectin or vWF expression. However, there were no differences in aggregation between ARC1779 and the placebo group in healthy volunteers. After perfusion, platelet activation was measured using blood from healthy volunteers in their research [38].

The use of aptamer in CAD patients
Our systematic literature search has indicated five studies on the use of aptamer to treat CAD. One study was conducted using an ex-vivo model (human blood), and the others used an in-vivo model (human).
Four studies used pegnivacogin and anivamersen reversal [35,[39][40][41]. According to a study by Posvic et al. [39], using these medications with at least 50% reversal demonstrated safety compared to the Heparin group. This RNA aptamer, pegnivacogin, is a secure anticoagulation method for invasively managed ACS patients. It was reported that its safety was likely due to the lower incidence of major bleeding and ischemic events because pegnivacogin is known as a selective factor IXa inhibitor [39]. The effect of pegnivacogin on activated platelets could be negated by ASO, anivamersen (RB007). In their study, Cohen et al. [40] also noted that an active reversal technique using RB007, followed by an anticoagulation strategy utilizing RB006 to suppress factor IXa, exhibits a balance of safety and efficacy following PCI.
Studies by Staudacher et al. and Chan et al. [35,41] used Pegnivacogin (RB006) as the active drug and RB007 as the antidote. Both approaches reduced the aggregation of thrombocytes in the pegnivacogin group compared to the placebo group. Pegnivacogin has been shown to decrease platelet aggregation in the blood of healthy individuals and in patients with acute coronary syndrome by inhibiting the formation of thrombin and lowering platelet reactivity, by indirect thrombin reduction. Pegnivacogin inhibits factor IXa by decreasing platelet activation and aggregation in vitro (Table 2) [35]. Chan et al. [41] showed that RB006 increased activated partial thromboplastin time in a dose-dependent manner, whereas RB007 reversed activated partial thromboplastin time to baseline values without serious side effects. The most common side effect was mucocutaneous bleeding, which occurred in 10% of participants.
The fourth study in the field of CAD indicated by our literature search involved the use of ARC1779 to examine its efficacy. ARC1779 has a rapid antiplatelet action. ARC1779 reaches maximum concentration level within 7-30 min [38]. The study by Arzamendi et al. demonstrated that giving ARC1779 as a first medication had an additive impact on the dual antiplatelet therapy by considerably lowering platelet adhesion in comparison to placebo [38]. Even in patients who had previously had aspirin and clopidogrel treatment, ARC1779 significantly decreased platelet adhesion but not platelet aggregation [38].

Definition and mechanism of action
Small interfering RNA (siRNA), silencing RNA or short interfering RNA, is a class of double-stranded (ds) RNA molecules with a length of about 20-25 nucleotides, which have various biological functions. One of its main roles is the RNA interference (RNAi) pathway, which destroys selective mRNAs by silencing or downregulating the expression of target genes [42].
RNAi's first step involves the cleavage of longer dsRNA molecules to shorter siRNAs, which typically have a dinucleotide overhang at the 3′ end of each strand. This process is mediated by an RNase III-like enzyme called Dicer. Once siRNAs are formed, they bind to a multiprotein complex called RISC (RNA-induced silencing complex). The siRNA strands then dissociate in the RISC complex and the more stable strand at the 5′ end usually integrates into the active RISC complex. The singlestranded antisense siRNA component is responsible for directing and targeting the RISC complex to the target mRNA. Finally, the mRNA is cleaved with the help of catalytic RISC proteins of the Argonaute family (Ago2) [43]. Table 3 illustrates the use of siRNA as therapy in studies of CVDs and risk factors, drug monitoring, and outcomes, including death, MACE, and allergic reactions.

The use of siRNA in healthy volunteers with elevated Lipoprotein(a)
Lipoprotein(a) has been previously known as an independent risk factor for atherothrombotic cardiovascular disease, which is genetically determined. Higher levels of Lp(a) have been linked in studies to an increased risk of stroke, myocardial infarction, and peripheral arterial disease (PAD) [44,45].
While some lipid-modifying treatments, such as niacin or PCSK9 inhibitors, have demonstrated moderate ability to reduce Lp(a) levels, no medication has yet been approved to treat elevated Lp(a) concentrations. The current study investigated different approaches to reduce Lp(a) levels, including the use of oligonucleotide therapies, among which SLN360 was included [44].
SLN360 is a specific kind of double-stranded, 19-nucleotide siRNA that has been covalently joined to a tri-antennary N-acetyl-galactosamine (GalNAc) moiety and chemically stabilized. The LPA gene, which codes for apolipoprotein(a) (apo[a]), a vital and rate-limiting component in the hepatic manufacture of the Lp(a) particle, has been specifically targeted by the treatment [44,45].
A study by Nissen et al. [44] investigated the effects of siRNA SLN360 on individuals who had elevated lipoprotein(a) levels but no prior history of cardiovascular disease. The results showed that SLN360 was generally well-tolerated, with only mild treatment-emergent adverse events reported, such as injection site reactions and headaches. None of these adverse events caused participant withdrawal. Of the 32 participants, only one experienced two serious adverse events, including hospitalization for post-SARS-CoV-2 vaccination headache and cholecystitis complications, which were considered unrelated to the study drug [44].   Collagen-induced aggregation: in healthy volunteers; averaged 18 Ω reduce to 2 Ω with abciximab, but unaffected by ARC1779 (18 Ω) in CAD patient; reduced to 3 Ω by abciximab, and unaffected by ARC1779 (4 Ω) Platelet activation This was done on blood samples from healthy volunteers after the perfusion experiments. Neither abciximab nor ARC1779 has a significant effect on P-selectin or vWF expression. Platelet− leukocyte binding increased after blood perfusion (control) compared with nonperfused blood (baseline), not significantly affected by ARC1779 or abciximab.
(continued on next page)       This study also demonstrated a dose-dependent decrease in plasma Lp

The use of siRNA in hypercholesterolemia and heterozygous familial hypercholesterolemia
High levels of low-density lipoprotein (LDL) cholesterol are important risk factors for the development of atherosclerotic cardiovascular disease. The risk of coronary heart disease changes by around 30% for every 30 mg/dL (0.78 mmol/L) variation in LDL cholesterol. Although statins have been proven to be effective in reducing LDL cholesterol levels, there is substantial variation in individual responses to these drugs, leading to the need for newer therapies [46][47][48].
Proprotein convertase subtilisin type 9 (PCSK9) is a newly identified target for lowering LDL cholesterol levels. Mutations leading to loss of function in PCSK9 are associated with lower circulating LDL cholesterol levels and reduced risk of cardiovascular disease [46,47]. Inclisiran (previously known as ALN-PCS) is an investigational, long-acting, chemically synthesized siRNA molecule directed against PCSK9 [47].
Inclisiran was shown to be safe in a phase I clinical trial in healthy volunteers, with all reported side effects being mild or moderate. No serious adverse events were drug-related or resulted in participant withdrawal from the study [46,47]. A phase II study evaluated the efficacy of subcutaneous inclisiran in patients at high cardiovascular risk and with elevated low-density lipoprotein cholesterol. The study found that inclisiran significantly decreased LDL cholesterol levels compared to a placebo. After a single dose of inclisiran, the least-squares mean reduction was 27.9-41.9%, and after two doses, it was 35.5-52.6% [48].
The safety and effectiveness of inclisiran were studied by Wright et al. [49] in individuals with varying degrees of renal impairment (RI). The study indicated that there were no discernible differences in the safety and pharmacodynamic effects of inclisiran in individuals with normal renal function when compared to those with mild, moderate, or severe RI. Inclisiran was found to significantly reduce PCSK9 levels and LDL-C in all renal function groups compared to a placebo (P < 0.001 vs. placebo in all groups). These findings indicate that inclisiran can be safely used in patients with RI without requiring any dose adjustments [49].
The phase III ORION-9 study evaluated inclisiran for the treatment of heterozygous familial hypercholesterolemia. A total of 482 adult patients participated in the study and were randomly assigned to receive subcutaneous injections of inclisiran sodium (300 mg) or placebo on days 1, 90, 270, and 450. By day 510, the research revealed that the inclisiran group had significantly lower LDL cholesterol levels than the placebo group, with percent changes of − 39.7% and 8.2%, respectively, and a between-group difference of − 47.9% points (P < 0.001). Inclisiran was effective in lowering LDL levels in all genotypes of FH, according to the results [9].

The use of siRNA in ASCVD and ASCVD risk equivalent
A study by Ray et al. (ORION-10) [50] demonstrated the effectiveness of inclisiran in lowering LDL cholesterol in patients with ASCVD. The study showed a significant reduction in LDL cholesterol levels compared to placebo with a mean placebo-corrected percentage change in LDL cholesterol levels of − 55.5% on day 90, -52.3% on day 510, and -54.9% on day 540 (P < 0.0001 for all). Nevertheless, adverse events at the injection site were more common in the inclisiran group than the placebo group, with a rate of 2.6% vs. 0.9%, respectively. All of these adverse events were mild and none were severe [50,51].
Inclisiran was studied by Raal et al. for safety and efficacy in South African patients who were at high risk of getting cardiovascular illnesses [9]. The data showed that inclisiran reduced LDL cholesterol levels in the South African patients while being both safe and effective. Inclisiran lowered LDL cholesterol levels by 54.2% compared to placebo, and the corresponding time-averaged reductions were 52.8% (P < 0.0001 for both) [9]. In the ORION-11 phase III study, inclisiran was found to have significant and sustained effects in reducing atherogenic lipoprotein concentrations in patients with ASCVD risk equivalent. The study involved 203 participants without a previous history of ASCVD, but with either type-2 diabetes mellitus, FH, or an expected 10-year risk of ≥20% for CVD. The participants were randomized to be given either a placebo or 300 mg of inclisiran sodium at specific intervals over 18 months. The percentage change in the concentration of LDL cholesterol from baseline was − 41.9% with inclisiran compared to +1.8% with the placebo, resulting in a − 43.7% difference between the groups (95% CI, − 52.8 to − 34.6; significant p-value) on day 510. Additionally, inclisiran was observed to significantly reduce non-HDL cholesterol and apoB compared to the placebo on day 510 [52]. A phase III pooled analysis of ORION-9, ORION-10, and ORION-11 examined inclisiran's potential benefits in reducing Major Adverse Cardiovascular Events (MACE). The MACE, including fatal and non-fatal incidences of myocardial infarctions and strokes, was lower in patients who received inclisiran than those who received placebo. Inclisiran significantly reduced composite MACE (OR 0.74, 95% CI 0.58-0.94), but not fatal and non-fatal myocardial infarctions (OR 0.80, 95% CI 0.50-1.27) or fatal and non-fatal strokes (OR 0.86, 95% CI 0.41-1.81) [51].

The use of siRNA in hATTR amyloidosis
Mutations in the transthyretin (TTR) gene lead to multisystem organ dysfunction in the rare and progressive condition known as hereditary transthyretin-mediated (hATTR) amyloidosis. Pathogenic TTR aggregation, misfolding, and fibrillization are the causes of amyloid buildup in numerous organs, including the peripheral nervous system and the heart. Our systematic literature search identified two siRNA treatments, Revusiran and patisiran, that are being investigated as potential therapies for hATTR amyloidosis [53,54].
Revusiran is a form of siRNA that specifically targets a section of the human TTR mRNA and is delivered to the liver, where TTR is mostly generated, by a triantennary N-acetylgalactosamine (GalNAc) ligand. Revusiran was well tolerated in phase I and phase II clinical trials and decreased serum transthyretin levels by up to 92.4%. This prompted the phase III ENDEAVOUR study, in which patients with hATTR amyloidosis and cardiomyopathy were enrolled. They were randomized in a 2:1 ratio to receive subcutaneous revusiran 500 mg (n = 140) or placebo (n = 66) daily for 5 days over the course of a week, followed by weekly doses [55].
In the revusiran phase III ENDEAVOUR study, there were more deaths in the revusiran group (18 out of 140 subjects, 12.9%) than in the placebo group (2 out of 66 subjects, 3.0%). The cause of the increased mortality associated with revusiran was investigated, but no definitive explanation was found, although revusiran could not be ruled out as a contributing factor. As a result, the study was stopped and further development of revusiran was discontinued [55].
A lipid nanoparticle (LNP) that makes it easier for the short interfering RNA to be transported to the hepatocytes, the main site of TTR, is included in patisiran, a type of RNAi therapy. By cleaving TTR messenger RNA once inside the cell, the small interfering RNA lowers the production of both mutant and wild-type TTR protein [56].
A phase I clinical study of patisiran by Coelho et al. [56] demonstrated proof of concept for RNAi therapy targeting messenger RNA. In a phase II open-label extension study in 2020, the long-term safety and tolerability of patisiran were assessed. One patient withdrew after roughly 19 months owing to gastro-esophageal cancer (which was assessed unlikely to be related to patisiran), and one patient died of myocardial infarction (not related to patisiran) after completing all doses but before the research's end. Of the 27 individuals who were initially enrolled, 25 patients finished the study. The most frequent drug-related side effects were mild flushing and infusion-related reactions, which occurred in 16 (59%) patients, and no patient discontinued the study as a result.
The effect of patisiran 0.3 mg/kg on individuals with polyneuropathy due to hATTR amyloidosis was examined in the APOLLO phase III trial. There were 225 patients total, 148 of whom received patisiran, and 77 who received a placebo. The modified Neuropathy Impairment Score+7 (mNIS+7) was changed from baseline after 18 months of treatment, according to the study. The results showed that the patisiran group had a decrease of − 6.0 ± 1.7 points, indicating less impairment, while the placebo group had an increase of +28.0 ± 2.6 points, indicating more impairment (mean difference, − 34.0 points; P < 0.001) [57].
A study by Obici et al. aimed to determine the effect of patisiran on the Quality of Life (QOL) of patients with hATTR amyloidosis. Many assessments were employed in the study, including the Norfolk Quality of Life-Diabetic Neuropathy (Norfolk QOL-DN), EuroQoL 5-dimensions 5-levels (EQ-5D-5L), EQ-VAS, Rasch-built Overall Disability Scale (R-ODS), and Composite Autonomic Symptom Score-31 (COMPASS-31). The results of the study are presented in a table and show that patients who received patisiran had better QOL across all measures compared to those who received placebo [58].
The efficacy of patisiran on a specific cardiac subpopulation from the APOLLO study participants was further analyzed by Minsamisawa et al. [59] and Solomon et al. [60]. Of 256 eligible patients, 126 (56%) did not have hypertension or aortic valve disease and had a baseline LV wall thickness of less than 13 mm. At 18 months, several cardiac parameters were observed and compared to placebo. In the cardiac subpopulation, it was discovered that patisiran dramatically reduced mean LV wall thickness, increased end-diastolic volume, decreased brain natriuretic peptide's N-terminal prohormone, and reduced overall longitudinal strain. These results imply that patisiran may slow or stop the progression of hATTR amyloidosis' cardiac symptoms.

Conclusion
RNA-based therapy has shown promising results in the management of CVDs and CV risk factors. However, data are still at an early stage and more studies are needed to address several challenges regarding the use of RNA-based therapy in clinical settings. Overall, either RNA-based therapy potentially revolutionizes cardiovascular disease therapy or provides a new avenue as an alternative therapeutic approach in the future.