RNA interference-based therapies for the control of atherosclerosis risk factors

INTRODUCTION

Atherosclerosis is a progressive chronic inflammatory disease leading to the accumulation of cholesterol containing plasma lipoproteins, smooth muscle cells, connective tissue and inflammatory cells in the vascular wall, causing coronary artery disease and predisposing to myocardial infarction, heart failure and stroke [1]. The development of atherosclerosis is a long process, in which both endogenous and exogenous factors have an impact on the disease progression. Healthy lifestyle is the basis of the primary prevention of atherosclerotic cardiovascular disease (ASCVD). However, several mutations and genetic changes have been recognized to increase the risk for atherosclerosis by elevating plasma levels of pro-atherogenic lipoproteins, like low-density lipoprotein (LDL), triglyceride-rich lipoprotein particles (TRLs) and lipoprotein (a) [Lp(a)], or by having an effect on pro-inflammatory activity and cell proliferation in the vascular wall [2,3]. Even though many of these targets have been discovered over 50 years ago, they have gained interest as potential targets for therapies only during the past ten years. In addition, due to increasing global incidence of obesity and type 2 diabetes, TRLs have created interest to be targeted for the treatment of ASCVDs [4,5▪]. Although current treatment strategies of atherosclerosis are almost exclusively focused on plasma lipid lowering and high blood pressure, the regulation of inflammatory response has also been considered as a target for novel therapeutic strategies [6,7]. Despite of the fast development of new therapies and the improved treatment strategies, ASCVDs are still the leading cause of death worldwide [8]. 

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RNA interference (RNAi) is a naturally occurring gene regulation mechanism, capable of silencing gene expression, which gives it a remarkable therapeutic potential. Current therapeutics utilizing RNAi include small interfering RNAs (siRNAs) that are complementary double-stranded RNA molecules, and antisense oligonucleotides (ASOs) that are single stranded RNA or DNA molecules [9,10]. Both siRNAs and ASOs bind to the target complementary messenger RNA (mRNA) and prevent the protein translation. Short hairpin RNAs (shRNA) have also been studied as potential tools for RNAi therapy, as they can be integrated into genome and are further processed into siRNAs, allowing more long-term knockdown of target mRNA [11].

RNAi technology, targeted to treat vascular inflammation and lipid metabolism, provides an effective therapeutic tool against ASCVDs. The first mRNA targeted therapy for homozygote familial hypercholesterolemia, mipomersen, was an ASO that targets apolipoprotein B-100 (ApoB-100) to reduce the level of LDL-C and Lp(a) [12]. However, the required high doses to reach the therapeutic effect elevated the risk of hepatotoxicity, resulting mipomersen being rejected by the European Medicines Agency (EMA), and in the United States it's usage is strictly restricted to patients with homozygous familial hypercholesterolemia [13,14]. The first-ever siRNA therapeutic, patisiran for the treatment of hereditary transthyretin-mediated amyloidosis, was approved by U.S. Food and Drug Administration (FDA) in 2018 and since then several others have proceeded to clinical trials [15]. The first siRNA therapy for the treatment of hypercholesterolemia, inclisiran, was approved by FDA not earlier than in 2021 [16▪]. It is worth noticing that current next-generation ASO therapies are N-acetylgalactosamine (GalNAc) conjugated which allows liver specific delivery and the use of lower doses to have therapeutic effects [17]. Ongoing coronavirus disease 2019 (COVID-19) pandemic and the success of nucleoside-modified mRNA vaccines have potentially broadened the landscape and scope of RNA therapies.

This review focuses on the status of RNAi-based therapies that currently are or have been in clinical development for ASCVD risk factors during past one and half years (Fig. 1). The review also addresses some potential future targets on resolving vascular inflammation that are currently at a preclinical stage.

F1FIGURE 1:

A graphical abstract. Summary of the current therapeutic targets and RNAi based therapies under clinical and preclinical development against ASCVD risk factors. ASCVD, atherosclerotic cardiovascular disease; RNAi, RNA interference.

RNA INTERFERENCE-BASED THERAPIES FOR LIPID LOWERING

High plasma cholesterol levels and adverse lipoprotein profiles with a high fraction of LDL, TRLs and Lp(a) are strong risk factors for ASCVDs and hereby in the main focus of drug development. Recent years have been invigorating for the development of RNAi-based therapies and new lipid lowering drugs have been enabled to enter the market. Novel lipid lowering pathways have been studied and new promising targets, such as angiopoietin-like protein 3 (ANGPTL3) and -4, apolipoprotein C-III (ApoC-III) and Lp(a) have advanced to clinical trials and shown promise for future therapies (Table 1).

Table 1 - Overview of ongoing RNAi-based clinical trials Target Drug name Phase Indication Approach Trial no. PCSK9 Inclisiran Phase III Homo- and heterozygote familiar hypercholesterolemia in adolescents GalNAc-siRNA NCT04659863NCT04652726 ApoC-III Volanesorsen, olezarsen Phase III Mixed dyslipidemia, severe hypertriglyceridemia and familial chylomicronemia syndrome GalNAc-ASO NCT05185843NCT05130450 ARO-APOC3 Phase II Mixed dyslipidemia, severe hypertriglyceridemia and familial chylomicronemia syndrome GalNAc-siRNA NCT04998201NCT04720534NCT05089084 ANGPTL3 Vupanorsen Phase II Hypercholesterolemia and hypertriglyceridemia GalNAc-ASO NCT03360747 ARO-ANG3 Phase II Mixed dyslipidemia and homozygote familial hypercholesterolemia GalNAc-siRNA NCT04832971NCT05217667 Lp(a) Pelacarsen Phase III ASCVDs, elevated serum Lp(a) GalNAc-ASO NCT04023552 Olpasiran Phase II ASCVDs, elevated serum Lp(a) GalNAc-siRNA NCT03626662NCT04270760

ASO, antisense oligonucleotide; ASCVD, atherosclerotic cardiovascular disease; GalNAc, N-acetylgalactosamine; Lp(a), lipoprotein (a); PCSK9, proprotein convertase subtilisin/kexin 9; siRNA, small interfering RNA; RNAi, RNA interference.


PROPROTEIN CONVERTASE SUBTILISIN/KEXIN 9

The first RNAi-based lipid lowering therapy, proprotein convertase subtilisin/kexin 9 (PCSK9) targeted GalNAc-modified siRNA inclisiran (Leqvio), was approved by EMA in late 2020 and by FDA in late 2021, for the treatment of heterozygote familial hypercholesterolemia, nonfamilial hypercholesterolemia and dyslipidemias in adults [16▪]. In addition, the combination therapy of inclisiran and other lipid-lowering therapies is indicated for the patients with no response to maximum tolerated dose of statins to alleviate hypercholesterolemia. Inclisiran very effectively lowers the plasma total cholesterol and LDL-C levels by reducing the expression of PCSK9 mRNA that decreases the degradation of LDL receptors and enhances liver uptake of LDL from plasma [18]. In a meta-analysis it caused the reduction of total cholesterol by 37%, LDL-C by 51%, ApoB by 41% and non-HDL-C by 45% [19]. In addition to the regulation of LDL metabolism, PCSK9 plays a role in the regulation of inflammatory response, as the inhibition of PCSK9 decreases the expression of vascular chemokines and adhesion molecules, making it also an attractive target to alleviate vascular inflammation and atherosclerosis [20].

A cardiovascular outcome study of inclisiran, ORION-11 (NCT03400800), resulted in >20% reduction in cardiovascular events, such as sudden death, myocardial infarction, and stroke in patients having ASCVD with risk factors like type 2 diabetes or familial hypercholesterolemia [21]. Two other clinical trials, ORION-13 (NCT04659863) and ORION-16 (NCT04652726), are still ongoing to investigate the safety, tolerability, and efficacy of inclisiran in homo- and heterozygous familial hypercholesterolemia in adolescents. Importantly, inclisiran has shown a very good safety profile, as no serious adverse events were reported in previous trials [19]. Inclisiran also provides long-term therapeutic effect, since it is dosed as an injectable formulation only after every 6 months. In comparison to other lipid lowering therapeutics, this relatively infrequent dosing could also help to overcome the challenges related to patient adherence, like fluctuations in LDL levels due to irregular treatment. However, cost-effectiveness of inclisiran has been a concern as previous, antibody based PCSK9 inhibitors have been relatively expensive, with the at annual cost around $5850 [22]. Novartis has announced the wholesale acquisition cost of inclisiran being $6500 in a year. In February 2021, the Institute for Clinical and Economic Review (ICER) published a report on value for inclisiran, recommending a price range of $3600–$6000 per year for it to be cost-effective [23]. Although the actual long term price tag of inclisiran is yet to be seen, it will likely be most cost-effective among higher risk patients who do not respond to other lipid lowering therapies and whose cholesterol levels remain on high level.

APOLIPOPROTEIN C-III AND ANGIOPOIETIN-LIKE PROTEIN 3

Besides the LDL particles, TRLs and especially their remnant particles have also proven to be pro-atherogenic and causally linked to cardiovascular diseases [5▪]. Clearance of these particles is regulated by several factors, but especially two proteins inhibiting lipoprotein lipase (LPL) activity and regulating triglyceride (TG) hydrolysis and TRL uptake, ApoC-III and ANGPTL3, have gained clinical interest. Albeit inclisiran is the only clinically approved siRNA for lipid lowering to date, several ASOs have been approved for the treatment of dyslipidemias. ApoC-III targeted ASO, volanesorsen, was approved by EMA in 2019 and several ApoC-III targeted ASOs are currently in clinical trials [24]. GalNAc-conjugated ASO, olezarsen (former known as AKCEA-APO-CIII-LRx), has shown promising results in a phase II clinical trial (NCT03070782), as it was well tolerated and resulted in a substantial reduction in fasting TG levels in patients with hypertriglyceridemia and a high risk for ASCVD [25▪]. There were no significant changes in LDL-C and total cholesterol, but in a phase III trial with familial chylomicronemia syndrome and hypertriglycemia (NCT02211209, NCT02300233) it provided a significant >70% reduction of TGs [26,27]. In addition, ARO-APOC-III ASO has entered phase II clinical trials (NCT04720534, NCT05089084, NCT04998201) for the treatment of mixed dyslipidemia, severe hypertriglyceridemia and familial chylomicronemia syndrome in 2021 [25▪,28]. In previous phase I trial, ARO-APOC-III treatment in healthy volunteers was well tolerated, and showed 70% reduction in TG levels, but also modest reduction in plasma LDL-C and 80% increase in plasma HDL levels.

Reduction of ANGPTL3 levels has been associated with lower plasma TGs and cholesterol levels in humans, both naturally with genetic loss-of-function mutations [29] and therapeutically [26,30,31]. Recently, ANGPTL3 was also shown to regulate the hepatic update of atherogenic particles in a manner independent of LPL regulation [32–34]. One of the latest suggested therapeutic agents targeting ANGPTL3 mRNA was vupanorsen, an ASO that selectively inhibits its expression in liver. In a phase II clinical trial published in 2020, subcutaneous dose of vupanorsen reduced patients ANGPTL3 by 62%, ApoC-III by 58% and total cholesterol by 19%, and led to a more favorable lipid profile [35]. However, in January 2022 Pfizer and Ionis Pharmaceuticals announced that they discontinued the clinical development of vupanorsen due to a too low magnitude of lipid reduction and its association with increased liver fat and elevated liver enzymes [36]. Nevertheless, another ANGPTL3 inhibiting clinical trial run by Arrowhead Pharmaceuticals and using a siRNA ARO-ANG3 for patients with mixed dyslipidemia is still ongoing (NCT04832971) and a new clinical trial for homozygote familial hypercholesterolemia has been recently started (NCT05217667).

Another therapeutic target for ASCVD from the angiopoietin-like protein family is ANGPTL4 that also inhibits LPL activity and modulates fatty acid metabolism, and is associated with atherosclerosis and type 2 diabetes [37,38▪]. Currently, the inhibition of ANGPTL4 via RNAi is at preclinical stage, but a study published July 2021 showed that the hepatic ANGPTL4 inhibition by GalNac-conjugated ANGPTL4-ASO protects mice against diet induced dyslipidemia and obesity [38▪].

LIPOPROTEIN (a)

High plasma Lp(a) level, which is mainly regulated by variations in the LPA gene coding for apolipoprotein (a), is an independent risk factor for ASCVDs [39]. It is also considered as a prognostic factor in patients after myocardial infarction [40]. Lp(a) is bound to apolipoprotein (a) as a part of LDL particle and Lp(a) levels can remain high and even increase despite of statin or PCSK9 inhibitor treatments, elevating the risk for cardiac events [41]. Thus, there is a demand for secondary prevention focused on lowering plasma Lp(a), to reduce the residual cardiovascular risk. Although there is significant methodological and patient dependent heterogeneity in the plasma Lp(a) levels, Lp(a ) levels over 50 mg/dl are generally considered elevated. From that, the risk of cardiac events increases stepwise, and Lp(a) over 100 mg/dl yields to two-fold higher risk of major adverse cardiovascular event when compared to individuals with Lp(a) below 10 mg/dl [42].

Currently, there are no approved therapies for high Lp(a) levels, but new RNAi-based therapeutics, pelacarsen, olpasiran and SLN360, have entered clinical development. Olpasiran is a GalNAc-conjugated siRNA targeting LPA mRNA and inhibiting the translation of Lp(a) [43▪]. Early phase I and II trials with oplasiran (NCT03626662, NCT04270760) have provided promising results in patients having very high Lp(a) levels. The reduction in Lp(a) levels in these trials was dose-dependent and up to 90% persisting for months after a single dose. In comparison to pelacarsen, the reduction of Lp(a) achieved with olpasiran was up to 72–80%, depending on the monthly or weekly dosing of the pelacarsen [39]. Pelacarsen (also known as IONIS-APO(a)-LRx , AKCEA-APO(a)-LRx , and TQJ230) is an ASO targeted to inhibit apolipoprotein(a) expression in hepatocytes [44]. The development of olpasiran and pelacarsen is still ongoing, and both therapies have shown a good tolerability and safety profile. Currently, a phase III clinical trial (NCT04023552) for further evaluation of the longer-term safety, tolerability, dosing, and effect on clinical outcomes of pelacarsen is going.

The newest therapeutic candidate for the Lp(a) lowering is a GalNAc-siRNA called SLN360. In a recent phase 1 clinical trial with 32 participants, SLN360 showed a dose-dependent lowering of plasma Lp(a) levels without major adverse effects [45]. A clinical trial for safety, dosage and pharmacokinetics of SLN360 started in late 2020 and it is currently recruiting (NCT04606602).

NOVEL INFLAMMATION-TARGETED MOLECULES FOR RNA INTERFERENCE THERAPY DEVELOPMENT

Despite the fast development of new therapies and improved treatment of plasma lipid lowering, ASCVDs are a global pandemic having a burgeoning demand for new treatment strategies and novel therapeutic agents. Three clinical trials, CANTOS, COLCOT and LoDoCo2, have revealed the large potential of anti-inflammatory drugs for ASCVDs, as the inhibition of interleukin (IL)-1β and anti-inflammatory compound colchicine reduced cardiovascular events in these trials [6,46]. However, these trials also met some challenges with impaired host defense, hilighting the need to target the possible new anti-inflammatory therapies to atherosclerotic lesions [47]. So far, the most attractive and promising therapeutic targets for resolving inflammation in atherosclerosis are NF-κβ-signaling pathway, transforming growth factor beta (TGF-β) and endothelial adhesion molecule P-selectin [1].

TGF-β is known to regulate inflammation and cell proliferation, differentiation and apoptosis, making it a potential target for therapies against diseases with a high inflammatory and cell cycle regulation component [48]. Endothelial TGF-β signaling is one of the driving agents of vascular inflammation, promoting lipid accumulation and atherogenesis [30]. In hyperlipidemic mice, TGF-β interfering siRNAs targeted to the vascular endothelium led to about 50% reduction of aortic lesions in comparison to control mice. When administrated after high fat diet, this 7C1-TGFβ nanoparticle therapy led to regression of already established atherosclerotic lesions in aorta, and reduction of arterial lipid burden in mice. These results suggest the potential of anti TGF-β therapies in the treatment of ASCVD, but due to the variety of TGF-β effects in the body, the safety and limitations of such therapies need to be closely evaluated. Optimally, treatment should be targeted to vascular endothelium, especially due to the previous mouse studies suggesting that the systemic TGF-β inhibition might promote atherogenic processes [49,50].

Another interesting RNAi based therapeutic agent targeting the vascular endothelium is a shRNA targeted to P-selectin adhesion molecules, leading to the down-regulation of the expression of Receptor for advanced glycation end products (RAGE) [51▪]. RAGE is a cell surface receptor that has been associated with chronic inflammation and atherosclerosis by binding multiple ligands and activating the pro-inflammatory NF-κB pathway [52]. A study published in 2021 showed that in ApoE-deficient mice, repeated intravenous administration of lipoplexes carrying Psel-lipo/shRAGE led to a reduction in RAGE expression in endothelial cells, reduction of serum inflammatory cytokines, and lower NF-κB and TNF-α protein levels in aorta [51▪]. Atherosclerotic lesions in the aortic root were also reduced by about 40% in Psel-lipo/shRAGE treated mice in comparison to the control animals.

Besides the shRNA mediated RAGE inhibition, there is novelty in the lipoplexes used as vectors [51▪]. Lipoplexes are synthetic nanoparticle complexes that are composed by binding the desired nucleic acid, for example therapeuric siRNA, with a lipid vesicle called liposome [53▪]. The equilibrium of these complexes can be disturbed for example by changes in pH, allowing the release of nucleic acid in the cells. Lipoplexes have not been clinically tested yet, but there is one FDA approved drug utilizing synthetic lipid nanoparticles (LNPs), for hereditary transthyretin amyloidosis. LNPs and lipoplexes share many similar features, but while lipoplexes are slightly bigger in size and formed by binding existing liposomes with nucleic acids, LNPs are rather formed in one step by embedding the nucleic acid with the lipid solution [54]. Currently, LNPs are also being used in two COVID-19 mRNA vaccines used worldwide, and there is an increasing interest in their use as tissue specific therapeutic vehicles for various diseases [55]. The P-selectin targeted lipoplexes used as carriers for Psel-lipo/shRAGE were reported being effectively taken up by LPS activated endothelial cells in vitro, and in vivo they accumulated in ApoE deficient mouse aortas without showing significant adverse effects [51▪]. Further studies will be needed to conclude whether this kind of lipoplexes could be efficient and safe tools for RNAi based therapies for atherosclerosis in humans.

ADVANTAGES AND FUTURE PROSPECTS OF RNA INTERFERENCE-BASED THERAPIES

The potential advantages that RNAi based therapeutics have over the conventional therapies for ASCVD include high specificity, less frequent dosing, and possibility to affect targets that cannot be modulated with conventional pharmaceuticals [56]. For example, siRNA-based medicine inclisiran provides therapeutic effect with just two injectable doses a year, and in the future higher potency shRNAs could allow even longer effects with lower dosing [19,57]. Noncoding RNA (ncRNA) biology is a new and promising field of medical research that is well suited for the development of RNAi-based therapies [58,59]. NcRNAs are classified in two groups, short microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), and these both ncRNAs have been considered as a ‘junk’ RNA for a long time, but are now gaining interest as promising therapeutic targets for several diseases, including ASCVDs. Targeting therapies to ncRNAs possess several beneficial effects, like high cell-type specificity that enables siRNAs or ASOs being delivered systemically but having an effect only on target tissue, minimizing the nonspecific side effects. Several ncRNAs have entered clinical trials during past years for the treatment of different diseases and disorders, but not yet for cardiovascular diseases.

However, harnessing the RNAi technologies for therapeutic use has not been straightforward, and their development has also faced challenges, like inefficient delivery, off-target effects and unwanted immune responses. In addition, nucleic acid degradation by endogenous nucleases has caused challenges, but various chemical modifications to RNAs have been developed and viral or nonviral vectors can be used as carriers to protect the RNA molecules and target the right tissues [53▪,56,60]. Chemical modifications are also used to avoid immune response activation and off-target effects of the RNAi therapy. The four already approved RNAi utilizing therapies for clinical use are very encouraging, but there is still a road ahead to overcome the challenges.

CONCLUSION

Current RNAi based therapies for atherosclerotic CVDs that have entered clinical trials are targeting plasma lipid levels and focusing on directing patient's lipoprotein profile to less atherogenic direction. However, there are some interesting preclinical studies targeting also vascular inflammation, along with interesting techniques for increased tissue specificity. Further research is needed to evaluate if RNAi based therapeutics could provide more targeted therapy with fewer side effects, and if they could be successfully and safely used along with statins to lower the residual risk of ASCVD.

Acknowledgements

Graphical abstract Created with BioRender.com.

Financial support and sponsorship

This work was supported by Finnish Foundation for Cardiovascular Research, Orion Research Foundation, Leducq Foundation, Academy of Finland Flagship program, and European Research Council.

Conflicts of interest

There are no conflicts of interest.

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

▪ of special interest

▪▪ of outstanding interest

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