The rapidly emerging field of RNA therapies for cardiovascular disease

In this issue, we focus on the bright future of ribonucleic acid (RNA) therapeutics which has unlimited potential as a platform for lifesaving and life-enhancing treatments, including RNA drugs directed against cardiovascular disease. RNA drugs can be broadly divided into coding or non-coding RNA drugs. Coding RNA includes messenger RNA (mRNA), self-amplifying RNA (saRNA), and circular RNA (circRNA)[1–2]. Non-coding RNA includes small interfering RNA (siRNA), microRNA (mirs), anti-microRNA (antagomirs), and aptamers[1–2]. Coding RNA provides a sequence that is translated by the ribosomal machinery of the cell into the encoded protein. Non-coding RNA, by virtue of its structure, may bind to proteins, deoxyribonucleic acid (DNA), or other RNA molecules, to affect their function in a therapeutic manner. Both coding and non-coding RNA drugs are now in clinical use, with many more in pre-clinical or clinical development. Most familiar to us is the RNA drug that has saved millions of lives in the past 5 years. The mRNA vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was highly successful, demonstrating extraordinary efficacy and safety, as well-documented by large clinical trials[3–4]. Almost 6 billion individuals have been vaccinated against SARS-CoV-2 worldwide, primarily with the mRNA vaccine, experiencing a very low adverse event rate compared to unvaccinated individuals, particularly those with pre-existing medical conditions[3–5]. The development of the vaccine was exceptionally rapid, with the first clinical trials initiated within 4 months of obtaining the sequence of the SARS-CoV-2 virus. The speed at which an RNA drug can be designed far surpasses that of other drug classes (small molecules, viral vectors, recombinant proteins), and the cost is at least an order of magnitude less. To be sure, these features contributed to the success of the SARS-CoV-2 vaccine. However, it is a less appreciated fact that years of work on the respiratory syncytial virus (RSV) identified mutations that stabilized class I fusion proteins to improve their immunogenicity[6]. This generalizable antigen design approach was quickly applied to the SARS-CoV-2 spike protein to make it an effective antigen. Thus, to be prepared for the next pandemic, it is critical to support the scientific investigation of vaccines against viral families that are likely to evolve into a pandemic. Vaccines can be designed for non-infectious diseases, such as cancer. Indeed, personalized neoantigen cancer therapy in early trials appears quite promising, and a novel way to address the great genetic variability of neoplasms[7–8]. With respect to cardiovascular disease, there are also opportunities to use mRNA vaccines to address pathophysiological processes in cardiovascular disease, as discussed by Zou et al.[9] in this issue. For example, one can generate vaccines that target factors regulating lipid metabolism (e.g., cholesteryl ester transfer protein) or cholesterol delivery to the vessel wall (e.g., apolipoprotein B) so as to facilitate a favorable lipid profile or improved vascular equilibrium to attenuate atherosclerosis. Lipid nanoparticles (LNPs) are necessary to protect coding RNA from degradation and to deliver the RNA to the cytoplasm where it can be translated into the encoded protein. It is well known that mRNA and saRNA lend themselves to rapid vaccine development. Exogenous RNA molecules, and the LNPs that act as their vehicle, are pro-inflammatory, and this property of the RNA/LNP drug can act as an adjuvant for the encoded vaccine[10]. Although this pro-inflammatory feature is beneficial for vaccines, it is negative for protein replacement therapies or gene editing applications. To a certain extent, the pro-inflammatory properties of mRNA and saRNA can be lessened by rigorous purification to remove impurities such as double-stranded mRNA[11]. In addition, the use of modified nucleotides such as 1-methyl-pseuduridine reduces recognition of the resulting modified mRNA (mmRNA) by pattern recognition receptors such as Toll-like receptor 3[11]. However, improved LNPs that are less pro-inflammatory and that target the cardiovascular system are also needed to advance mRNA cardiovascular therapeutics. Furthermore, most LNPs are taken up by the liver (ionizable lipids) or the spleen and lung (cationic lipids). The lipids used in generating the nanoparticle can be modified to modestly reduce uptake in the liver, lung, and spleen. Alternatively, antibodies or aptamers can be used to decorate the surface of the LNP to target a specific cell type. In this issue, Rurik et al.[12] describe one intriguing cardiovascular application where LNPs were decorated with an antibody against CD5, to target T effector cells. The LNPs carried a mmRNA encoding a chimeric antigen receptor against FAP, a protein expressed by activated myofibroblasts. When infused systemically into a murine model of cardiac fibrosis, the LNP/mmRNA generated CAR-T cells in vivo, against activated myofibroblasts in the heart, and reduced cardiac fibrosis. Rurik et al.[12] describe a variety of other mRNA molecules that have promise in modulating the immune system, including mRNA encoding cytokines, signaling molecules, or epigenetic factors. Another approach to target the cardiovascular system is to design an mRNA that is only expressed in cardiovascular cells. In this issue, Adjmi et al.[13] describe their specific modRNA translation (SMRT) system to target cardiomyocytes. In essence, this drug is a double construct system, with the first construct expressing a therapeutic protein, and the second construct expressing an inhibitory protein designed to accelerate the degradation of the first construct (which contains a recognition sequence for the inhibitory protein). The second construct contains a seed sequence for microRNA that is preferentially expressed in the cardiomyocytes. Thus, in the heart, these microRNA bind to the second construct, accelerating its degradation, and preventing the translation of the inhibitory protein. Thus, the first construct is now free from inhibition and is translated into the therapeutic protein selectively in cardiomyocytes. Such a system might be used to provide mmRNA drugs that enhance cardiomyocyte proliferation, facilitate cardiomyocyte protection from ischemic injury, and/or induce cardiovascular regeneration[13]. The preferential uptake of LNPs by the liver is advantageous for many disorders of metabolism, including dyslipidemias. Underway are two clinical trials of an mRNA/LNPs for patients with heterozygous familial hypercholesterolemia[14–15]. In one trial, the LNP carries an mRNA encoding a Cas9/adenine deaminase fusion protein (a base editor) directed against the gene encoding pro-protein subtilisin kinase 9 (PCSK9). The base editor makes a single-base (A-to-G) change to inactivate the PCSK9 gene[14]. Because PCSK9 binds to and increases the degradation of the hepatic low-density lipoprotein (LDL) receptor, a non-functional PCSK9 permits increased LDL receptor expression. The increase in hepatic LDL receptor increases hepatic clearance of LDL cholesterol and reduces its serum levels. This is a gene therapy that causes a long-term reduction in serum LDL cholesterol with a single treatment[14]. A similar trial is underway with the base editor complex targeting ANGPTL3[15]. Reducing the expression of this enzyme also reduces serum LDL cholesterol. As mRNA and saRNA therapies are advanced to the clinic for cardiovascular diseases, improvements in manufacturing processes will improve the yield of high-quality products. In this issue of the Journal, Li et al.[16] study a major cause of abortive mRNA transcripts (transcripts that are less than full length). Typically, mRNA or saRNA drug substances are generated from linear DNA templates using in vitro transcription (IVT). Thus, the sequence design of the linear DNA templates containing the gene of interest (GOI) plays a critical role in the quality of mRNA therapeutics. A conserved sequence motif ATCTGT was previously identified as an endogenous T7 RNA polymerase (RNAP) class II pause site. Pausing of the polymerase can precipitate its dissociation from the DNA template, leaving behind a truncated RNA transcript. In the current study, Li et al.[16] describe versions of the motif that also serve as pause sites. Furthermore, they found that the position of the T7 RNAP pause site in the open reading frame determined the likelihood of premature termination. When the T7 RNAP pause site was more distant from the initiation site, more abortive transcripts were observed. The investigators also demonstrate that when DNA templates are observed to generate abortive transcripts, a search for T7 RNAP pause sites should be made. Subsequently, non-synonymous mutation of the pause site can generate a template that is transcribed into mRNAs with improved integrity, purity, and mRNA yield[16]. Another form of RNA that encodes protein is circRNA[17]. Because circRNA lacks ends, it is not susceptible to exonucleases. Thus, it persists longer in a cell than does mRNA, and generates protein for a longer duration. An internal ribosome entry site (IRES) permits translation of the encoded protein, and may also be modified to provide for preferential tissue expression. The circRNA is also less immunogenic than mRNA and does not require modified nucleosides[17]. For these reasons, there is much enthusiasm about circular RNA for generating therapeutic proteins. In one study, circRNA had greater persistence than mmRNA in endothelial cells[18]. Furthermore, circRNA encoding human telomerase (hTERT) was more effective than mRNA hTERT in reversing endothelial senescence[18]. In endothelial cells derived from patients with Hutchison-Gilford Progeria Syndrome, circRNA hTERT had a greater benefit than mRNA hTERT on telomere length, DNA damage, mitochondrial function, intercellular communication, and other measures of cell function and cell identity[18]. Such a circular RNA product may have broad applications as endotheliopathy contributes to most cardiovascular diseases, vascular dementia, radiation injury, inflammatory and autoimmune diseases, and other disorders of conduit vessels and the microvasculature. Circular RNA is fast approaching the clinic because of its intrinsic advantages as a coding RNA. Indeed, the first clinical trial of a circRNA product was initiated this March by the Shanghai company RiboX[19]. Their circRNA encodes the water channel aquaporin. It will be delivered to the salivary glands of patients whose tissues have been damaged by radiotherapy for head and neck cancer, to treat dry mouth syndrome. Although there may be greater familiarity with coding RNA drugs, such as mRNA vaccines, the first RNA drugs to be approved for cardiovascular indications were non-coding RNA drugs, as well discussed by Rurik et al.[12] in this issue. Among these is inclisaran, which is approved for use in patients with heterozygous familial hypercholesterolemia not responsive to maximal statin therapy[20]. Inclisaran is an siRNA that binds to the endogenous mRNA encoding PCSK9, thereby reducing the expression of this protein, increasing the LDL receptor, and lowering serum LDL cholesterol. The siRNA drug patisiran reduces the expression of transthyretin, benefiting patients with transthyretin-mediated amyloidosis and neuropathy, and may also have application in treating cardiac amyloidosis due to this disease[21]. Rurik et al.[12] provide a comprehensive discussion of the role of non-coding RNAs (e.g., lncRNA, miRNA, enhancer RNA) in immune regulation of cardiovascular disease, and the opportunities for targeting these RNA species with therapeutic microRNA sponges, antisense oligomers, or mmRNA[12]. Another class of non-coding RNAs in clinical trials for cardiovascular indications is the aptamers. Aptamers are DNA or RNA-based constructs, with modifications that reduce their degradation and markedly increase their stability in vivo, and structures that give them high affinity for their targets. The first aptamer to have a cardiovascular indication was Macugen, an anti-angiogenic agent targeting vascular endothelial growth factor[22]. This agent inhibited choroidal neovascularization and was approved for use in age-related macular degeneration. One of the pioneers of aptamer development for cardiovascular indications is Dr. Bruce Sullenger of Duke University. In this issue, Dr. Sullenger describes an aptamer targeting factor IXa (FIXa)[22]. The binding of the aptamer to FIXa prevents its activity, acting as an anticoagulant. In a rat model of extracorporeal membrane oxygenation, he and his colleagues compared the aptamer to the commonly used anticoagulant heparin, finding it to be similar in its anti-thrombotic effect. One advantage of the aptamer is that it has less effect on the platelet count as does heparin. Either drug can cause bleeding, in which case, the anticoagulant effect must be reversed. Heparin is reversed by protamine. Protamine can, in rare cases, cause an anaphylactic reaction. The anticoagulant effect of the FIXa aptamer can be reversed by a matching aptamer that binds with great affinity and does not share the side effects of protamine[22]. Thus, the FIXa aptamer may be particularly useful in patients with heparin-induced thrombocytopenia, and warrants clinical development. To conclude, RNA may be thought of as biological software. The code for this software may be rapidly designed, manufactured, and tested, and its applications are only limited by our imagination. Fundamental insights about the pathophysiology of a disease can be directly translated into new therapeutic molecules that re-constitute a signaling pathway or a deficient protein in the case of coding RNA; or non-coding RNA molecules that antagonize or activate a biological process by virtue of their interaction with protein, DNA, or other RNA molecules. The relatively simple manufacturing process of small batches of mRNA lends itself to individualized treatments, such as personalized neoantigen cancer therapy. It also lends itself to the development of deployable manufacturing units that can supplement manufacturing needs in low- and middle-income countries, or global manufacturing needs during a pandemic. These deployable manufacturing units will also permit academic medical centers or small biotech companies to provide point-of-care personalized RNA therapeutics. For these reasons, we are at the dawn of a new era in drug development, delivery, and distribution that will change the way we practice medicine, and will provide solutions for the many scourges that afflict us, including cardiovascular disease. AUTHOR CONTRIBUTIONS JPC is the sole author of this paper, and wrote and revised drafts of this article. CONFLICTS OF INTEREST STATEMENT John P. Cooke is the Honorary Editor of Cardiology Plus. DATA SHARING STATEMENT Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.