RNA Therapeutics for Cardiovascular Disease

Abstract

Purpose of Review:

The development of mRNA vaccines against COVID-19 has brought worldwide attention to the transformative potential of RNA-based therapeutics. The latter is essentially biological software that can be rapidly designed and generated, with an extensive catalog of applications. This review aims to highlight the mechanisms of action by which RNA-based drugs can affect specific gene targets and how RNA drugs can be employed to treat cardiovascular disease, with the focus on the therapeutics being evaluated in clinical trials. The recent advances in nanotechnology aiding the translation of such therapies into the clinic are also discussed.

Recent Findings:

There is a growing body of studies demonstrating utility of RNA for targeting previously “undruggable” pathways involved in development and progression of cardiovascular disease. Some challenges in RNA delivery have been overcome thanks to nanotechnology. There are several RNA-based drugs to treat hypercholesterolemia and myocardial infarction which are currently in clinical trials.

Summary

RNA therapeutics is a rapidly emerging field of biotherapeutics based upon a powerful and versatile platform with a nearly unlimited capacity to address unmet clinical needs. These therapeutics are destined to change the standard of care for many diseases, including cardiovascular disease.

Introduction

The field of RNA therapeutics has recently garnered worldwide attention due to its role in the first two vaccines for the COVID-19 virus. However, this is just one example of the therapeutic revolution that is underway. The promise of RNA derives from the fact that it is essentially biological software that can be rapidly designed and generated, with an extensive catalog of applications based on its modulation of the expression or activity of any clinically relevant protein. RNA-based therapeutics allows for a wide variety of applications, as manifested by ongoing clinical development of vaccines for infectious disease and for cancer, for enzyme replacement, and for modification of cell therapy. There are also unlimited possibilities for application for cardiovascular diseases [1]. The translation of RNA biology into therapies is recently made possible by new advances in the synthesis, design, and delivery methods for nucleic acid therapies.

RNA mechanisms of action

Messenger RNAs (mRNAs) are the transient blueprints of genes encoded in the genomic DNA [2]. The mRNA transcripts are acted upon by the translational machinery of the cell to generate the encoded proteins[3,4]. RNA-based therapeutics can be designed to introduce an exogenous mRNA into the cell [3,5]or they may be developed to inhibit the translation of endogenous mRNA. [6,7].

Silencing of endogenous mRNA can be achieved by several means, including antisense oligonucleotides (ASO), small interfering RNAs (siRNA), and microRNAs (miRNA) (Figure 1). Although all these RNAs bind to their complementary sequences on target mRNA and prevent its translation, the molecular pathways employed by each to interfere with endogenous mRNA are different. Therapeutic ASOs are short single-stranded nucleic acids that form a duplex with the target mRNA, recruiting RNase H to hydrolyze the mRNA. Alternatively, the RNA/ASO duplex can sterically block pre-mRNA processing and mRNA translation [8–10]. By contrast, endogenous siRNA and miRNA are non-coding RNA duplexes that must be processed by cell machinery and interact with RNA-induced silencing complex (RISC), which ultimately cleaves mRNA or represses its translation [11–15]. siRNA is entirely complementary to the target transcript and therefore affects the expression of a single gene (highly specific), whereas miRNA is partially complementary and may affect multiple genes. Agomirs and antagomirs are second generation small RNAs for modulating RNA expression that are highly stable when administered in vivo. Agomirs are chemically modified double-strand miRNA mimics with a modified antisense strand having phosphorothioates at the 5′ and 3′ ends, 3′ end cholesterol group, and nucleotide 2′-methoxy modification along the full length of the construct. Agomirs behave like mature endogenous miRNA and silence their target mRNA. Antagomirs have a similar structure but constitutively inhibit the activity of target miRNAs. These improvements in design of nucleic acid therapeutics have enhanced stability ( use of 2′-OMe and phosphorothioate modifications); and delivery (e.g. cholesterol conjugation to enhance distribution and cell permeation) [16]. Because the mRNA-silencing strategy can be designed to inhibit the translation of mRNA encoding any protein, they are in some cases superior to conventional pharmacotherapies. Specifically, they can therapeutically alter the expression of proteins that are otherwise not easily affected by small molecules, e.g. proteins that have an inaccessible conformation or non-enzymatic function [17].

Figure 1. Mechanisms of Action for RNA Therapeutics.

miRNA and siRNA use the RISC (RNA-induced silencing complex) to degrade the target gene’s complementary mRNA strands or to prevent protein translation, thereby downregulating the expression of a given target gene. Antisense Oligonucleotides (ASOs) rely on various mechanisms to inhibit translation of a target mRNA sequence, which may include steric blocking and ribonuclease H decay of pre-mRNA. Conversely, therapeutic mRNA accesses the cytoplasmic translational machinery to express a protein that is useful in treating a disease. Silencing the expression of endogenous mRNA has proved useful in the treatment of hypercholesterolemia, while expression of target proteins has proven effective in the vaccines for COVID-19, and of promise in the treatment of myocardial infarction (AZD8601 - VEGF). Created with BioRender.com

On the other hand, mRNA therapeutics supply an exogenous message which is introduced into the cell to generate a therapeutic protein, e.g. to replace a defective protein (gene replacement therapy) or introduce transcripts not naturally present in the cell (antigens for vaccination, chimeric antigen receptors for cell therapy). As opposed to silencing of endogenous mRNA which typically requires constructs in the range of tens of nucleotides, therapy with mRNA typically utilizes RNA constructs with thousands of nucleotides, resulting in substantial differences in the synthesis of these two classes of molecules. Nevertheless, mRNA therapeutics can be designed to encode virtually any protein or antigen and can thus also address “undruggable targets”. RNA sequences can be easily modified, allowing for personalization of mRNA therapy. Because the mRNA is translated into protein in the cytoplasm, and does not enter the nucleus it may be more feasible and safer than DNA-based gene therapy (plasmids, viral vectors) which must enter the cell nucleus, thereby posing a risk of integration into the host genome [18].

The therapeutic effect of synthetic mRNA was first demonstrated in 1992 when mRNA encoding vasopressin injected in the hypothalamus of rats with central diabetes insipidus transiently reversed the disease [19]. Although manufacturing of RNA therapeutics is relatively simple and involves either chemical synthesis (ASOs, siRNA, microRNA)[20] or enzymatic transcription in vitro (IVT) from the DNA template (mRNA)[21], early attempts to use RNA as a drug were challenged by its intrinsic ability to activate innate immune signaling and cause inflammation and apoptosis [22,23]. This hurdle was overcome by generating a number of chemically modified nucleotides and incorporating them into an RNA molecule [24]. Furthermore, it was demonstrated that some byproducts generated during IVT, such as double-stranded RNA, may contribute to inflammatory activation. This observation stimulated development of advanced purification methods, which, along with chemical modifications, dramatically reduced RNA toxicity [25]. In addition, advances in RNA design helped to enhance RNA stability and translatability through sequence optimization (both UTRs and open reading frame)[26–28]. A recently proposed approach to circularize mRNA also reduces RNA fragility, as it becomes resistant to degradation by ubiquitously present RNA exonucleases [29,30]. In addition, some lyophilization protocols were tested to prolong RNA shelf life and simplify its transportation and storage[31,32]. All aforementioned advances have made mRNA clinically promising in the last decade. However, delivery remains a hurdle for RNA-based cardiovascular therapeutics and requires special consideration.

Cardiovascular Delivery of RNA Therapeutics

Preclinical studies have shown proof-of-concept for the use of adeno-associated virus (AAV; Figure 2) serotype 9 which preferentially delivers miRNA to the cardiac myocytes, for example to enhance myocyte proliferation after myocardial infarction [33]. However, there are inherent shortcomings of viral vectors, such as an immune response to the vector. To overcome this obstacle, lipid nanoparticles have begun to replace viral vectors as the chosen delivery method for RNA-based drugs[34] (Figure 2) due to their favorable safety profile, low immunogenicity, and ease of manufacture. To be sure, lipid nanoparticles (LNPs) are not entirely inert and innocuous, as demonstrated by the rare anaphylactic reactions to the RNA vaccines, which have been attributed to the PEG component of the LNPs [35].

Figure 2. Delivery Platforms for nucleic acid therapeutics.

RNA often requires a delivery vehicle to be injected. One approach to deliver nucleic acid therapies are viral vectors, such as adeno-associated virus. A non-viral approach is to use liposome encapsulation using nanoparticles. These nanoparticles are typically self-assembled lipid vesicles surrounding the nucleic acids. This latter strategy has been used for Pfizer and Moderna COVID-19 vaccines and treatments for myocardial infarction currently in clinical trials. Created with BioRender.com

In an example of non-viral delivery, Khan and colleagues inhibited aberrant endothelial expression of Tie2 siRNA delivered systemically to nonhuman primates using a polymeric nanoparticle. This approach successfully reduced Tie2 expression in the heart, lungs, retina, kidney with minimal toxicity [36]. However, in this case, the siRNA would be expected to be delivered to normal and diseased endothelium. Recent advances in lipid nanoparticle systems can target activated endothelium, such as that overlying the atherosclerotic plaque [37,38].

Aside from localized delivery of drug eluting stents [39,40], successful strategies for delivery have focused on exploiting differences in blood flow characteristics within the vasculature and alterations to the endothelium to target diseased tissue. In this regard, blood flow throughout the arterial tree usually is laminar with shear stress in the range of 10–20 dynes/cm². Endothelial cells are responsive to shear stress, by virtue of mechanoreceptors, flow-activated ion channels, and associated proteins such as Yes-associated protein (YAP) and its paralog transcription activator with PDZ binding motif (TAZ) that regulate cell structure and functions such as expression and activity of nitric oxide synthase and vascular endothelial growth-factor receptor [41,42].

At bends, branches and bifurcations, as well as at sites of vascular disease, disturbed flow generates a low oscillatory shear stress upon the endothelium and alters signaling pathways, including BMP–TGFβ, WNT, Notch, HIF1α, TWIST1 and NFkB, contributing to the expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule[43], as well as changes in permeability [44]. These changes perpetuate a cycle of vascular inflammation, but also provide a pathway to target treatments specifically to these areas.

One strategy that leverages the endothelial adhesiveness of diseased vessels is the generation of leukocyte-based biomimetic nanoparticles (i.e., leukosomes). Leukosomes are assembled by integrating membrane proteins from leukocytes into the lipid nanoparticle (Figure 3). The leukosomes target activated endothelium and exhibit superior accumulation in atherosclerotic vessels as well as tumor vessels in mouse models [38].

Figure 3. RNA Liposome Structure and Surface Modifications.

The basic structure of a lipid RNA particle consists of phospholipids and cationic phospholipids and RNA. The cationic (net positive charge) of some phospholipids drives interaction with the negative phosphate backbone of RNA, effectively encapsulating it; other lipids complete the liposome formation in a structure commonly known as a “lipoplex.” At the surface, liposomes can be functionalized with various modifications that alter their distribution throughout the body. Some of these modifications include polymer coating of the surface lipids (e.g., polyethylene glycol or PEG), the inclusion of membrane or recombinant proteins (for targeting or transmigration) in the bilayer, and covalent modifications with fluorophores for imaging. Created with BioRender.com

In a seminal study, leukosomes were synthesized using membrane proteins purified from activated J774 macrophages, and loaded with rapamycin[37]. In Apo E −/− mice, leukosomes localized to the diseased aorta to a substantially greater degree than standard lipid nanoparticles. Furthermore, macrophage proliferation was reduced to a greater degree, and correlated with a greater decrease in levels of MCP (monocyte chemoattractant protein)-1 and IL (interleukin)-b1 in the aortic wall, as well as reduced aortic MMP (matrix metalloproteinases) activity, in mice treated with rapamycin using leukosomes by comparison to standard LNP delivery. This proof-of-concept showed that the leuko-rapa platform could suppress macrophage proliferation within the aorta after a short dosing schedule (7 days) with a favorable toxicity profile [37]. This treatment could be a promising intervention for the acute stabilization of late-stage plaques, and while this study used rapamycin as a cargo load, as a lipid nanoparticle, the formulation can be adjusted to encapsulate RNA.

Rather than directly targeting inflamed vasculature, Krohn-Grimberghe and colleagues used lipid nanoparticles that accumulate specifically within the bone marrow together to deliver siRNA inhibiting monocyte chemotactic protein 1 (MCP1). Using this platform, they decreased leukocyte release from the hematopoietic niche and, in a mouse model, improved healing after infarction while attenuating heart failure[45]. Thus, cardiovascular disease might also be treated by targeting other organs, rather than the heart or blood vessels directly. In fact, as discussed below, current RNA therapies for cardiovascular disease are largely targeted to the liver, taking advantage of the property of untargeted lipid nanoparticles to be taken up by the reticuloendothelial system in the liver and spleen.

The future of RNA therapies for cardiovascular disease is likely headed in the direction of targeted therapeutics as evidenced in the novel approaches using nanoparticles that localize to activated endothelium. A biomimetic approach, recapitulating adhesion and transmigration through the functionalization of the LNP with membrane proteins has proof-of-concept. Lessons from these studies will likely beget more research into related biomimetic approaches to replicate leukocyte adhesion to generate the second generation of RNA-LNPs for cardiovascular disease.

RNA therapeutics currently in clinical trials

Despite significant advances in treatment options, cardiovascular disease remains the number one cause of death in the United States and is responsible for 17.8 million deaths worldwide [46]. While there are only a handful of clinical studies for RNA therapeutics for cardiovascular diseases, the increased research and investment in this area ensure that there will be more of these studies in the coming years.

One of the earliest nucleic acid therapies for cardiovascular disease was Mipomersen, a second-generation phosphorothioate antisense oligonucleotide (ASO) targeting mRNA encoding apolipoprotein B-100. The drug was initially approved for treating homozygous familial hypercholesterolemia. Mipomersen reduced total LDL levels[47] without increasing total reactive C-protein levels[48]. Despite these results, Mipomersen was discontinued from clinical use in 2013 by the European Medicines Agency (EMA) in Europe over severe liver toxicity [49,50]. Based on the results, later generations of ASOs have been chemically modified to have reduced toxicity. In a recent phase II trial of TQJ230 (AKCEA-APO(a)-LRx), an ASO targeting lipoprotein(a) mRNA, more than 90% of patients achieved lipoprotein(a) concentrations below 50 mg/dl, after either 20 mg weekly injection or 60 mg injection every four weeks [51]. Another ASO that has reached clinical trials is volanesorsen. This ASO can be injected subcutaneously and functions by binding APOC3 mRNA and promoting its degradation. In a phase II study, volanesorsen, decreased Apoc-II (−80%) and triglycerides (−71%), and increased HDL-C levels (46%)[52]. Initial discouraging results with ASOs have not deterred research in this area, and newer iterations have been modified chemically [53] to reduce toxicity.

Another approach to lowering cholesterol is to target mRNA encoding the enzyme proprotein convertase subtilisin/kexin type 9 (PCSK9) which is predominantly produced in the liver [54–56]. PCSK9 binds to the low-density lipoprotein receptor (LDL-R) on the surface of hepatocytes, leading to the degradation of LDL-R, thereby increasing plasma LDL-cholesterol (LDL-C) levels. Inclisaran is a synthetic, chemically modified siRNA targeting PCSK9 mRNA with a covalently attached triantennary N-acetylgalactosamine (GalNAc) ligand. Inclisiran has met all primary and secondary endpoints across three phase III trials, had a favorable safety profile, and matched the LDL-lowering efficiency of antibody-based PCSK9 inhibitors alirocumab (Praluent; Regeneron/Sanofi) and evolocumab (Repatha; Amgen) after a twice-annual subcutaneous injection [51,57,58]. In patients with severe hypercholesterolemia on maximal doses of anti-lipid therapies (e.g. statins), inclisaran reduce LDL cholesterol by 40–50%. On the basis of these studies, inclisaran has been approved by the European Commission for the treatment of drug-resistant hypercholesterolemia in adults.

Beyond drugs inhibiting endogenous mRNA, the first and only cardiovascular-related mRNA drug to have reached clinical trials, is the mRNA encoding VEGF-A mRNA (Moderna). In a phase I study, intradermal administration of VEGF-A mRNA led increased local VEGF-A protein expression (as assessed by cutaneous microdialysis) and increased skin blood flow in men with type 2 diabetes [59]. Based on these results, a phase 2a clinical trial, the EPICCURE, will determine if this mRNA therapeutic restores ischemic but viable myocardial regions in patients with coronary artery disease, as assessed by ejection fraction [60]. This is a randomized, double-blind, placebo-controlled, multicenter study in patients with moderately impaired systolic function undergoing coronary artery bypass surgery. Patients are randomized to doses of 0, 3 or 30mg of VEGF A mRNA in a citrate buffer by epicardial injections (dosage split into 30 injections delivered into the ischemic myocardium, as pre-operatively mapped by PET imaging). If this programmatic effort is successful, it would provide evidence that direct injection of mRNA into an ischemic tissue may improve perfusion and function. A caveat is that many prior angiogenic gene therapies have failed, perhaps because of incomplete knowledge regarding the appropriate dose, duration, and delivery of the angiogenic agents [61].

Conclusion

The growth of RNA therapeutics has been facilitated by high throughput screening techniques to find target genes and sequences [62,63], which discoveries have been complemented by novel modifications of the RNA constructs to improve stability and to reduce toxicity; as well as improved delivery methods such as lipid nanoparticles [64]. A critical conjunction of advances is enabling the promise of RNA therapeutics, which includes the ability to create new drugs for “undruggable” targets; to rapidly generate vaccines against infectious diseases underlying pandemics; to truly personalize therapy. The promise of RNA therapeutics is as limitless as the genetic code on which it is based.

Recognizing the importance of this new therapeutic arena, in our hospital we have built a fully-integrated RNA Therapeutics program that supports our internal scientists, as well as external academic groups or small companies that hope to translate their basic research insights toward a transformative therapy. We perform fundamental research in mRNA sequence and structure underlying stability and translation. We have a cGMP program with clean rooms for the synthesis, purification, and validation of RNA constructs. We work with our Nanomedicine colleagues to generate suitable lipid nanoparticle constructs for local and systemic delivery of mRNA constructs. We have a Comparative Medicine Program with expertise in GLP preclinical studies; a first-in-man clinical trials unit for Phase 1 and 2a studies; and a large hospital system with a clinical research infrastructure that supports industry-sponsored Phase 2 and 3 clinical trials. Furthermore, we have an industry sponsor for manufacturing large batches of mRNA constructs for Phase 2, 3 and commercialization. Essentially, we have created an assembly line for academic research groups and small companies that hope to translate their mRNA constructs into transformative therapies. Our fully integrated hospital-based RNA therapeutics program provides a single-entry point with consultation to ensure a seamless development and translation of RNA-based drugs into the clinic. The need for such program was dictated by the fact that many small groups with innovative ideas for disruptive mRNA therapeutics lack key translational competencies to reach the clinic. To our knowledge, we are the only academic group with the infrastructure to fully support small academic groups and biotech startups, and in the coming years, we hope to support internal and external translational efforts to attain the great promise of RNA Therapeutics.

KEY POINTS.

• The therapeutic application of RNA has recently become a reality due to advances in the design, synthesis, purification, and delivery methods for RNA.

• RNA-based drugs can target previously “undruggable” pathways by silencing expression of proteins involved in pathogenesis or by providing an exogenous message to generate a therapeutic protein.

• RNA-based therapies are at an early stage of application in cardiovascular disease, but results are encouraging for hypercholesterolemia (with PCSK9 RNA-silencing) and myocardial infarction (with exogenous VEGF mRNA) Increased interest and investment in this area will ensure that there will be more RNA drug candidates in the coming years.

• Recognizing the importance of this new therapeutic arena, our group has built a fully-integrated hospital-based RNA therapeutics program to support internal and external scientists that hope to translate their basic research insights toward a transformative therapy.