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

RNA-targeted siRNA Therapeutics—Development of RNA Interference Strategies for Cardiovascular Disease

Discovery and Mechanisms of RNA Interference

The Nobel price-winning discovery of RNAi—potent and specific gene silencing by double-stranded RNA—was rapidly explored and exploited not only for basic research but was also successfully translated into clinical studies. Its adaptation for clinical translation—requiring a very high safety level—was technically demanding but could be achieved thanks to breakthroughs in nucleic acid chemistry and was further improved by novel ways to enhance in vivo delivery and targeting (e.g. GalNac-conjugation). Figure 3 illustrates the rapid pace of these developments.

Figure 3.

History of RNAi and CRISPR in the context of genome research. Against the history of genetic research, the very recent discovery of novel cellular mechanisms (e.g. RNAi, CRISPR-Cas) has led to unusually rapid progress towards their therapeutic utilization. Before their possible entry into the clinical arena, however, critically important safety issues need to be addressed.

RNAi may be triggered by chemically synthesized siRNAs, which are investigated in clinical trials already (Table 1), or recombinant shRNAs which are currently of experimental interest. siRNAs use the molecular apparatus of the RNA-induced silencing complex (RISC), a ribonucleoprotein complex containing an Argonaute (Ago) protein.

Delivery Strategies for siRNA Therapeutics

Multiple strategies have been developed to achieve selective targeting of nucleic acids drugs. The currently most advanced approach is GalNac coupling of the respective nucleic acid, which is then targeted to asialoglycoprotein receptors (ASGPR) on hepatocytes, that is substantially increasing the potency of the respective drug as described above for ASOs. There is currently no similarly efficient non-viral system in vivo for the targeting of other cell types (cardiomyocytes, immune cells) that are also of high cardiovascular interest.[39–45]

From the clinical perspective (Figure 4), GalNac-coupling enables a most convenient way of simple subcutaneous injection of the siRNA, which is subsequently delivered via bloodstream through the hepatic fenestrated epithelium to the ASGP receptors on the hepatocytes. Moreover, recent clinical trials have revealed that upon receptor-mediated cellular entry of advanced siRNAs which are chemically engineered to ablate immunogenicity and enhance stability, their resulting complex with RISC (Figure 2) is highly stable. This results in long-term (up to >6 months) efficient cleavage of the siRNA's target transcripts and accordingly suppression of the respective encoded protein. From the patient's perspective, this type of siRNA application may resemble a 'vaccination' due to the rather long-term effect of the procedure regarding, e.g. the patient's LDL-C level.

Figure 4.

Practical application of ASO and siRNA therapeutics. Whereas the development of the novel nucleic acid-based drugs (ASOs, siRNAs, anti-miRs) for in vivo use is technically most demanding, their clinical application may be surprisingly simple under certain circumstances. In particular the liver emerged as a relatively handy target, after the successful development of delivery systems based on the GalNac (ligand)- ASGPR (receptor) interaction. This has enabled efficient and specific drug delivery upon subcutaneous injection. In sharp contrast, the heart remains to constitute a hard target which needs to be addressed using invasive procedures.

Cardiovascular Translational Trials and Their Pharmaceutical Strategies

siRNA for PCSK9. Currently far advanced is the ORION clinical trials programme, where RNAi-mediated PCSK9 inhibition is investigated in diverse CVD cohorts with regard to clinical efficacy and safety (Table 1 and Note added in proof). The results of the ORION-1 trial (NCT02597127) have been published.[4,5,46–50] Patients who received the siRNA drug Inclisiran experienced profound LDL-C reduction by targeting PCSK9. Of note, persistent reduction of both PCSK9 and LDL-C after a single drug dose over a period of 240 days suggests that RNAi-mediated PCSK9 inhibition in the liver represents an alternative to mAb-targeting of circulating PCSK9 and almost certainly involves a lower injection burden. The phase-3 ORION-10 and ORION-11 studies (including >3.000 patients) confirmed efficacy for LDL lowering and safety (see Note added in proof). The ORION-4 study is an ongoing phase III clinical cardiovascular outcomes trial.

siRNA for Transthyretin. TTR amyloidosis has also been addressed by an siRNAi drug. In the APOLLO phase-III clinical trial (NCT01960348), the anti-TTR sRNAi drug patisiran improved multiple clinical manifestations of hereditary TTR amyloidosis with polyneuropathy.[51] In a subanalysis of this trial, patisiran improved left ventricular global longitudinal strain as compared to placebo.[52,53]

Extreme Versatility of Therapeutic RNA Structures

It is important to note that, fundamentally different from DNA, RNAs are carrying information not only in their linear sequences of nucleotides (primary structure), but local nucleotide pairing creates secondary structures, e.g. hairpins, and interactions among distantly located sequences create tertiary structures.[54–57] In fact, this structural versatility needs to be considered for RNAs as therapeutic tools as well as targets. The plethora of RNA types, sequences, and structures created by evolution is a treasure trove of potential therapeutic tools and targets[58,59] which is just beginning to be explored. Whereas the molecular mode of action of siRNAs has already been investigated in-depth, the biological functions of the abundantly expressed long non-coding RNAs (lncRNAs),[60–72] circular RNAs (circRNAs),[73–77] and multiple other naturally occurring[58,78–87] or purely synthetic (aptamers, spiegelmers)[88–93] RNA species are largely unknown. Through pathways that are conserved in eukaryotes from yeast to humans, diverse types of small RNAs (endo- and exo-siRNAs, primary and secondary piRNAs) are capable to direct cellular molecular machinery to silence gene expression.[78] Beyond the exogenous siRNAs in current use, other small synthetic RNAs may help to expand the versatility and programmability of RISCs for therapeutic purposes.[78]