From Traditional Pharmacological Towards Nucleic Acid-based Therapies for Cardiovascular Diseases

Ulf Landmesser; Wolfgang Poller; Sotirios Tsimikas; Patrick Most; Francesco Paneni; Thomas F. Lüscher


Eur Heart J. 2020;41(40):3884-3899. 

In This Article

Recent Developments of RNA-targeted Antisense Oligonucleotide Therapeutics for Cardiovascular Disease

Molecular Mechanisms and Delivery of ASO Therapeutics

Advanced nucleic acid chemistry has enabled the development of purely synthetic 'nucleic acid drugs' which may retain key structural features of RNA or DNA but also are profoundly modified (e.g. 'xeno nucleic acids', XNAs). Sophisticated recent methods of nucleic acid chemistry have allowed development of clinically safe and efficient 'antisense' drugs, i.e. synthetic oligonucleotides which provide target gene silencing.[13] ASOs are generally 13–20 nucleic acids long and use Watson-Crick hybridization to bind to the target RNA. However, unmodified, naked DNA or RNA used as drugs are easily degraded and are not efficacious. To overcome this limitation, modifications of the backbone (replacing P-O with P-S moieties) and/or sugar moieties, particularly at the 2′ position with a variety of chemistries, have increased both the affinity for the cognate sequence and also enhanced resistance to enzymes that degrade these drugs, resulting in higher potency, improved tolerability and clinical effectiveness.

Additional recent advances include specific targeting and delivery of ASOs to hepatocytes by conjugation of N-acetylgalactosamine (GalNAc) to ASOs that bind to high capacity asialoglycoprotein receptors on the liver, thereby improving potency up to 30-fold as compared to unconjugated molecules.[7] ASOs are designed as 'gapmers', with the middle 10 bases as DNA that mediate RNAse H1 cleavage of the sense strand, and the wings at either side as modified DNAs to enhance stability of binding, potency, and tolerability. Single-stranded ASOs bind directly to messenger RNA (mRNA) to create a duplex that is then acted upon by RNAse H1 present in every cell to mediate target mRNA cleavage and prevent protein production (Figure 2). The antisense strand is relatively resistant to cleavage and can then bind to another target mRNA, resulting in long half-lives of these drugs (3–4 weeks). This allows for both lower dose to achieve any required effect and also prolonged dosing frequency. Supplementary material online, Figure S1 provides an overview of the key strategies and methodological breakthroughs towards the development of clinically applicable ASO drugs Suppl. Table S1.[14–20]

Suppl. Figure S1.

Important Cardiovascular Clinical Trials With RNA-targeted ASOs

Novel lipid-lowering ASO therapeutics are either recently approved or under regulatory review/clinical development (Table 1), including ASOs targeting ApoC-III, lipoprotein (a) [Lp(a)], and angiopoietin-like3 (ANGPTL3).[6,7,10,21–23] All three are addressing molecular targets which were so far inaccessible for efficient treatment by conventional pharmacotherapy approaches.

ASOs Targeting ApoC-III. Volanesorsen is a non-GalNAc second generation ASO that was approved in the EU for familial chylomicronemia syndrome (FCS).[21] Such patients have elevated dietary-derived chylomicrons in plasma due to either lipoprotein-lipase (LPL) deficiency or related enzymes and generally exhibit recurrent bouts of acute pancreatitis and are at increased risk of ACVD later in life. It was demonstrated that apoC-III is both an inhibitor of LPL but also delays clearance of triglyceride-rich lipoproteins, thus volanesorsen is effective in FCS through the latter mechanism.[22] Volanesorsen is highly effective in lowering triglyceride levels by 78%, achieving triglyceride levels below the pancreatitis threshold in most patients. Both FCS and volanesorsen can each be associated with thrombocytopenia but with regular monitoring of the platelet count there have been no major bleeding episodes. A GalNac-conjugated ASO targeted to ApoC-III with the same sequence as volanesorsen is in late stages of phase 2 development in patients with prior history of CVD and triglycerides ≥200 mg/dL in patients with pre-existing CVD is ongoing (NCT03385239). A phase 1 trial in volunteers with elevated triglycerides, including with monthly dosing, showed broad improvement in the atherogenic lipid profile, including significant reductions in triglycerides, total cholesterol, ApoB, non-high-density lipoprotein cholesterol (HDL-C), very low LDL cholesterol (LDL-C), and increases in HDL-C with a favourable safety and tolerability profile.[24] The results of these trials will determine the design of a phase 3 cardiovascular outcomes trial in patients with elevated triglycerides and pre-existing CVD.

ASOs Targeting Apolipoprotein(a). ASO treatment targeting apolipoprotein(a) has also advanced to a phase 3 cardiovascular outcomes trial, with the study design being recently reported (NCT04023552) and a start date January 2020. The Lp(a) HORIZON trial is planned to recruit 7680 subjects with prior cardiovascular events [myocardial infarction (MI), stroke, or peripheral artery disease] and Lp(a) >70 mg/dL (~>175 nmol/L). They will be randomized to TQJ230 80 mg s.c. monthly vs. placebo for a median of 4 years. The study is designed as a superiority trial with two primary endpoints: (i) time to first occurrence of major adverse cardiovascular events (cardiovascular death, non-fatal MI, non-fatal stroke, and urgent coronary Revascularization requiring hospitalization) as confirmed by the adjudication committee in patients with elevated Lp(a) ≥70 mg/dL and (ii) time to the first occurrence of clinical endpoint of major adverse cardiovascular events in a population of patients with elevated Lp(a) ≥90 mg/dL. The clinical development leading to this trial included a phase 1 and a phase 2 trial with the non-GalNAc parent molecule, and a phase 2 GalNAc molecule with similar nucleic acid sequence in which the 6 P-S groups had been removed from the backbone.[6,7]

GalNAc conjugation and additional structural modifications in the study drug IONIS-APO(a)-LRx resulted in >30-fold higher potency with >80% reduction in Lp(a) levels. Furthermore, the reductions in Lp(a) were accompanied by significant reductions in proinflammatory OxPL, LDL-C, and apoB-100. Of note, the promigratory behaviour of circulating monocytes to endothelial cells was attenuated while the participants received the drug, whereas monocytes regained their promigratory phenotype thereafter. The extent of Lp(a) lowering with a single dose once monthly should be sufficient to allow normalization of Lp(a) concentrations in almost all patients.

ASOs Targeting ANGPTL3. ANGPTL3 is a protein synthesized primarily in the liver. Its key physiological role is to inhibit LPL and endothelial lipase. As such ANGPTL3 is a novel, genetic target for CVD risk reduction. Patients with complete loss of function in ANGPTL3 have familial hypolipidaemia, that is characterized by life-long, very low levels of LDL-C, triglycerides, and HDL-C.[25] Importantly, loss of function mutations in ANGPTL3 are also associated with reduced CVD risk.[26] In a phase 1 study of human volunteers with elevated triglyceride levels, a GalNAc-modified ASO has been reported to mimic the lipid phenotype of patients with familial combined hypolipidaemia.[26] In animal models, it reduced liver triglyceride content and atherosclerosis progression while increasing insulin sensitivity. Importantly, ANGPTL3 inhibition also reduces very LDL-C (27.9–60.0%), non-high-density lipoprotein cholesterol, ApoB, and ApoC-III, suggesting that it may represent an ideal drug to treat patients with elevated remnant cholesterol despite optimal lipid modifying therapy otherwise. A phase 2 study is currently underway (NCT03371355).

ASOs Targeting Transthyretin. Transthyretin amyloidosis can, in addition to the nervous system, also affect the heart resulting in severe cardiomyopathy.[1–3] The 2′-MOE-engineered ASO inotersen, which acts by reducing the production of both wild-type and mutant TTR in the liver, improved the disease course and quality of life in hereditary TTR amyloidosis polyneuropathy in a 15-month phase-III study.[1] Pilot studies suggest that it may also improve cardiac function in patients with cardiac involvement.[27–29] Inotersen is currently approved worldwide for use in TTR polyneuropathy. A GalNac version is also in phase I trials and is planned to be developed for both polyneuropathy and hereditary and wild-type TTR cardiac amyloidosis (NCT03728634).

ASOs Targeting Dystrophin. ASOs have also been investigated for the clinical treatment of Duchenne muscular dystrophy (DMD).[30–33] Cardiomyopathy occurs in a substantial fraction of DMD patients,[34] but exon-skipping ASO technology has so far been evaluated in animal models only.[35–38]