Modified mRNA-Mediated CCN5 Gene Transfer Ameliorates Cardiac Dysfunction and Fibrosis without Adverse Structural Remodeling.

Modified mRNAs (modRNAs) are an emerging delivery method for gene therapy. The success of modRNA-based COVID-19 vaccines has demonstrated that modRNA is a safe and effective therapeutic tool. Moreover, modRNA has the potential to treat various human diseases, including cardiac dysfunction. Acute myocardial infarction (MI) is a major cardiac disorder that currently lacks curative treatment options, and MI is commonly accompanied by fibrosis and impaired cardiac function. Our group previously demonstrated that the matricellular protein CCN5 inhibits cardiac fibrosis (CF) and mitigates cardiac dysfunction. However, it remains unclear whether early intervention of CF under stress conditions is beneficial or more detrimental due to potential adverse effects such as left ventricular (LV) rupture. We hypothesized that CCN5 would alleviate the adverse effects of myocardial infarction (MI) through its anti-fibrotic properties under stress conditions. To induce the rapid expression of CCN5, ModRNA-CCN5 was synthesized and administrated directly into the myocardium in a mouse MI model. To evaluate CCN5 activity, we established two independent experimental schemes: (1) preventive intervention and (2) therapeutic intervention. Functional analyses, including echocardiography and magnetic resonance imaging (MRI), along with molecular assays, demonstrated that modRNA-mediated CCN5 gene transfer significantly attenuated cardiac fibrosis and improved cardiac function in both preventive and therapeutic models, without causing left ventricular rupture or any adverse cardiac remodeling. In conclusion, early intervention in CF by ModRNA-CCN5 gene transfer is an efficient and safe therapeutic modality for treating MI-induced heart failure.

Direct reprogramming induces vascular regeneration post muscle ischemic injury.

Reprogramming non-cardiomyocytes (non-CMs) into cardiomyocyte (CM)-like cells is a promising strategy for cardiac regeneration in conditions such as ischemic heart disease. Here, we used a modified mRNA (modRNA) gene delivery platform to deliver a cocktail, termed 7G-modRNA, of four cardiac-reprogramming genes-Gata4 (G), Mef2c (M), Tbx5 (T), and Hand2 (H)-together with three reprogramming-helper genes-dominant-negative (DN)-TGFß, DN-Wnt8a, and acid ceramidase (AC)-to induce CM-like cells. We showed that 7G-modRNA reprogrammed 57% of CM-like cells in vitro. Through a lineage-tracing model, we determined that delivering the 7G-modRNA cocktail at the time of myocardial infarction reprogrammed ∼25% of CM-like cells in the scar area and significantly improved cardiac function, scar size, long-term survival, and capillary density. Mechanistically, we determined that while 7G-modRNA cannot create de novo beating CMs in vitro or in vivo, it can significantly upregulate pro-angiogenic mesenchymal stromal cells markers and transcription factors. We also demonstrated that our 7G-modRNA cocktail leads to neovascularization in ischemic-limb injury, indicating CM-like cells importance in other organs besides the heart. modRNA is currently being used around the globe for vaccination against COVID-19, and this study proves this is a safe, highly efficient gene delivery approach with therapeutic potential to treat ischemic diseases.

The matricellular protein CCN5 prevents adverse atrial structural and electrical remodelling.

Atrial structural remodelling including atrial hypertrophy and fibrosis is a key mediator of atrial fibrillation (AF). We previously demonstrated that the matricellular protein CCN5 elicits anti-fibrotic and anti-hypertrophic effects in left ventricles under pressure overload. We here determined the utility of CCN5 in ameliorating adverse atrial remodelling and arrhythmias in a murine model of angiotensin II (AngII) infusion. Advanced atrial structural remodelling was induced by AngII infusion in control mice and mice overexpressing CCN5 either through transgenesis (CCN5 Tg) or AAV9-mediated gene transfer (AAV9-CCN5). The mRNA levels of pro-fibrotic and pro-inflammatory genes were markedly up-regulated by AngII infusion, which was significantly normalized by CCN5 overexpression. In vitro studies in isolated atrial fibroblasts demonstrated a marked reduction in AngII-induced fibroblast trans-differentiation in CCN5-treated atria. Moreover, while AngII increased the expression of phosphorylated CaMKII and ryanodine receptor 2 levels in HL-1 cells, these molecular features of AF were prevented by CCN5. Electrophysiological studies in ex vivo perfused hearts revealed a blunted susceptibility of the AAV9-CCN5-treated hearts to rapid atrial pacing-induced arrhythmias and concomitant reversal in AngII-induced atrial action potential prolongation. These data demonstrate the utility of a gene transfer approach targeting CCN5 for reversal of adverse atrial structural and electrophysiological remodelling.

Role of the PRC2-Six1-miR-25 signaling axis in heart failure.

The reduced expression of cardiac sarco-endoplasmic reticulum Ca2+ ATPase (SERCA2a) is a hallmark of heart failure. We previously showed that miR-25 is a crucial transcriptional regulator of SERCA2a in the heart. However, the precise mechanism of cardiac miR-25 regulation is largely unknown. Literatures suggested that miR-25 is regulated by the transcriptional co-factor, sine oculis homeobox homolog 1 (Six1), which in turn is epigenetically regulated by polycomb repressive complex 2 (PRC 2) in cardiac progenitor cells. Therefore, we aimed to investigate whether Six1 and PRC2 are indeed involved in the regulation of the miR-25 level in the setting of heart failure. Six1 was up-regulated in the failing hearts of humans and mice. Overexpression of Six1 led to adverse cardiac remodeling, whereas knock-down of Six1 attenuated pressure overload-induced cardiac dysfunction. The adverse effects of Six1 were ameliorated by knock-down of miR-25. The epigenetic repression on the Six1 promoter by PRC2 was significantly reduced in failing hearts. Epigenetic repression of Six1 is relieved through a reduction of PRC2 activity in heart failure. Six1 up-regulates miR-25, which is followed by reduction of cardiac SERCA2a expression. Collectively, these data showed that the PRC2-Six1-miR-25 signaling axis is involved in heart failure. Our finding introduces new insight into potential treatments of heart failure.

miR-25 Tough Decoy Enhances Cardiac Function in Heart Failure.

MicroRNAs are promising therapeutic targets, because their inhibition has the potential to normalize gene expression in diseased states. Recently, our group found that miR-25 is a key SERCA2a regulating microRNA, and we showed that multiple injections of antagomirs against miR-25 enhance cardiac contractility and function through SERCA2a restoration in a murine heart failure model. However, for clinical application, a more stable suppressor of miR-25 would be desirable. Tough Decoy (TuD) inhibitors are emerging as a highly effective method for microRNA inhibition due to their resistance to endonucleolytic degradation, high miRNA binding affinity, and efficient delivery. We generated a miR-25 TuD inhibitor and subcloned it into a cardiotropic AAV9 vector to evaluate its efficacy. The AAV9 TuD showed selective inhibition of miR-25 in vitro cardiomyoblast culture. In vivo, AAV9-miR-25 TuD delivered to the murine pressure-overload heart failure model selectively decreased expression of miR-25, increased levels of SERCA2a protein, and ameliorated cardiac dysfunction and fibrosis. Our data indicate that miR-25 TuD is an effective long-term suppressor of miR-25 and a promising therapeutic candidate to treat heart failure.

Enhancing atrial-specific gene expression using a calsequestrin cis-regulatory module 4 with a sarcolipin promoter.

BACKGROUND: Cardiac gene therapy using the adeno-associated virus serotype 9 vector is widely used because of its efficient transduction. However, the promoters used to drive expression often cause off-target localization. To overcome this, studies have applied cardiac-specific promoters, although expression is debilitated compared to that of ubiquitous promoters. To address these issues in the context of atrial-specific gene expression, an enhancer calsequestrin cis-regulatory module 4 (CRM4) and the highly atrial-specific promoter sarcolipin were combined to enhance expression and minimize off tissue expression. METHODS: To observe expression and bio-distribution, constructs were generated using two different reporter genes: luciferase and enhanced green fluorescent protein (EGFP). The ubiquitous cytomegalovirus (CMV), sarcolipin (SLN) and CRM4 combined with sarcolipin (CRM4.SLN) were compared and analyzed using the luciferase assay, western blotting, a quantitative polymerase chain reaction and fluorescence imaging. RESULTS: The CMV promoter containing vectors showed the strongest expression in vitro and in vivo. However, the module SLN combination showed enhanced atrial expression and a minimized off-target effect even when compared with the individual SLN promoter. CONCLUSIONS: For gene therapy involving atrial gene transfer, the CRM4.SLN combination is a promising alternative to the use of the CMV promoter. CRM4.SLN had significant atrial expression and minimized extra-atrial expression.

Inhibition of miR-25 ameliorates cardiac and skeletal muscle dysfunction in aged mdx/utrn haploinsufficient (+/-) mice.

Dystrophic cardiomyopathy is a significant feature of Duchenne muscular dystrophy (DMD). Increased cardiomyocyte cytosolic calcium (Ca2+) and interstitial fibrosis are major pathophysiological hallmarks that ultimately result in cardiac dysfunction. MicroRNA-25 (miR-25) has been identified as a suppressor of both sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) and mothers against decapentaplegic homolog-7 (Smad7) proteins. In this study, we created a gene transfer using an miR-25 tough decoy (TuD) RNA inhibitor delivered via recombinant adeno-associated virus serotype 9 (AAV9) to evaluate the effect of miR-25 inhibition on cardiac and skeletal muscle function in aged dystrophin/utrophin haploinsufficient mice mdx/utrn (+/-), a validated transgenic murine model of DMD. We found that the intravenous delivery of AAV9 miR-25 TuD resulted in strong and stable inhibition of cardiac miR-25 levels, together with the restoration of SERCA2a and Smad7 expression. This was associated with the amelioration of cardiomyocyte interstitial fibrosis as well as recovered cardiac function. Furthermore, the direct quadricep intramuscular injection of AAV9 miR-25 TuD significantly restored skeletal muscle Smad7 expression, reduced tissue fibrosis, and enhanced skeletal muscle performance in mdx/utrn (+/-) mice. These results imply that miR-25 TuD gene transfer may be a novel therapeutic approach to restore cardiomyocyte Ca2+ homeostasis and abrogate tissue fibrosis in DMD.

Modified mRNA Formulation and Stability for Cardiac and Skeletal Muscle Delivery.

Directly injecting naked or lipid nanoparticle (LNP)-encapsulated modified mRNA (modRNA) allows rapid and efficient protein expression. This non-viral technology has been used successfully in modRNA vaccines against SARS-CoV-2. The main challenges in using modRNA vaccines were the initial requirement for an ultra-cold storage to preserve their integrity and concerns regarding unwanted side effects from this new technology. Here, we showed that naked modRNA maintains its integrity when stored up to 7 days at 4 °C, and LNP-encapsulated modRNA for up to 7 days at room temperature. Naked modRNA is predominantly expressed at the site of injection when delivered into cardiac or skeletal muscle. In comparison, LNP-encapsulated modRNA granted superior protein expression but also additional protein expression beyond the cardiac or skeletal muscle injection site. To overcome this challenge, we developed a skeletal-muscle-specific modRNA translation system (skeletal muscle SMRTs) for LNP-encapsulated modRNA. This system allows controlled protein translation predominantly at the site of injection to prevent potentially detrimental leakage and expression in major organs. Our study revealed the potential of the SMRTs platform for controlled expression of mRNA payload delivered intramuscularly. To conclude, our SMRTs platform for LNP-encapsulated modRNA can provide safe, stable, efficient and targeted gene expression at the site of injection.

Optimization of Synthesis of Modified mRNA.

Modified mRNA (modRNA) is a safe and effective vector for gene-based therapies. Notably, the safety of modRNA has been validated through COVID-19 vaccines which incorporate modRNA technology to translate spike proteins. Alternative gene delivery methods using plasmids, lentiviruses, adenoviruses, and adeno-associated viruses have suffered from key challenges such as genome integration, delayed and uncontrolled expression, and immunogenic responses. However, modRNA poses no risk of genome integration, has transient and rapid expression, and lacks an immunogenic response. Our lab utilizes modRNA-based therapies to promote cardiac regeneration following myocardial infarction and heart failure. We have also developed and refined an optimized and economical method for synthesis of modRNA. Here, we provide an updated methodology with improved translational efficiency for in vitro and in vivo application.

Matricellular Protein CCN5 Gene Transfer Ameliorates Cardiac and Skeletal Dysfunction in mdx/utrn (±) Haploinsufficient Mice by Reducing Fibrosis and Upregulating Utrophin Expression.

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration due to dystrophin gene mutations. Patients with DMD initially experience muscle weakness in their limbs during adolescence. With age, patients develop fatal respiratory and cardiac dysfunctions. During the later stages of the disease, severe cardiac fibrosis occurs, compromising cardiac function. Previously, our research showed that the matricellular protein CCN5 has antifibrotic properties. Therefore, we hypothesized that CCN5 gene transfer would ameliorate cardiac fibrosis and thus improve cardiac function in DMD-induced cardiomyopathy. We utilized mdx/utrn (±) haploinsufficient mice that recapitulated the DMD-disease phenotypes and used an adeno-associated virus serotype-9 viral vector for CCN5 gene transfer. We evaluated the onset of cardiac dysfunction using echocardiography and determined the experimental starting point in 13-month-old mice. Two months after CCN5 gene transfer, cardiac function was significantly enhanced, and cardiac fibrosis was ameliorated. Additionally, running performance was improved in CCN5 gene-transfected mice. Furthermore, in silico gene profiling analysis identified utrophin as a novel transcriptional target of CCN5. This was supplemented by a utrophin promoter assay and RNA-seq analysis, which confirmed that CCN5 was directly associated with utrophin expression. Our results showed that CCN5 may be a promising therapeutic molecule for DMD-induced cardiac and skeletal dysfunction.

Conventional Method of Transverse Aortic Constriction in Mice.

Transverse aortic constriction is a widely used surgical model to reflect the progression from cardiac hypertrophy to heart failure states due to left ventricular pressure overload in mice. It produces afterload increase on the left ventricle in which compensated hypertrophy initially occurs in the first 2 weeks. This develops into maladaptive remodeling of the left ventricle and atrium, leading to heart failure. This model is useful for cardiac studies since transverse aortic constriction can be consistently replicated and has low surgical mortality. Additionally, the gradual progression to cardiac failure makes it a valuable method to evaluate the efficacy of potential therapeutic intervention. We introduce this chapter to offer practical approaches to facilitate a simple methodology for transverse aortic constriction.

Generation of Efficient miRNA Inhibitors Using Tough Decoy Constructs.

Over the last decade a previously unappreciated mechanism of gene regulation has been uncovered that is mediated by a large class of small noncoding RNAs known as microRNAs (miRNAs), and this mechanism is utilized by organisms ranging from plants to humans. MiRNAs are important downregulators of gene expression and are seen to be dysregulated in disease development. Thus inhibition of aberrantly upregulated miRNAs as a therapeutic approach has become a promising field.Many models of miRNA inhibitors currently exist, with decoy models being the most successful in current research. A promising inhibition model is the tough decoy (TuD) RNAs inhibitor, which uses antisense sequences to bind to target miRNAs, preventing them from binding to their endogenous targets. Since the TuD inhibitors have the ability to be successfully used in vitro and in vivo studies, this is a covetable inhibition method. In this chapter, we introduce how to design and generate miRNA tough decoy inhibitors with an adeno-associated viral construct. TuD inhibitors will have two miRNA binding sites. The TuD will include stem sequences, a miRNA binding site, and linkers. In vitro validation experiments to confirm the effectiveness of the TuD to inhibit miRNA are described. We also propose some practical approaches for making a TuD for miRNA of interest. We hope this chapter facilitates readers to create a simpler method to generate TuD that can be used for miRNA loss of function studies.

Cytokine-Like 1 Regulates Cardiac Fibrosis via Modulation of TGF-ß Signaling.

Cytokine-like 1 (Cytl1) is a secreted protein that is involved in diverse biological processes. A comparative modeling study indicated that Cytl1 is structurally and functionally similar to monocyte chemoattractant protein 1 (MCP-1). As MCP-1 plays an important role in cardiac fibrosis (CF) and heart failure (HF), we investigated the role of Cytl1 in a mouse model of CF and HF. Cytl1 was upregulated in the failing mouse heart. Pressure overload-induced CF was significantly attenuated in cytl1 knock-out (KO) mice compared to that from wild-type (WT) mice. By contrast, adeno-associated virus (AAV)-mediated overexpression of cytl1 alone led to the development of CF in vivo. The endothelial-mesenchymal transition (EndMT) and the transdifferentiation of fibroblasts (FBs) to myofibroblasts (MFBs) have been suggested to contribute considerably to CF. Adenovirus-mediated overexpression of cytl1 was sufficient to induce these two critical CF-related processes in vitro, which were completely abrogated by co-treatment with SB-431542, an antagonist of TGF-ß receptor 1. Cytl1 induced the expression of TGF-ß2 both in vivo and in vitro. Antagonizing the receptor for MCP-1, C-C chemokine receptor type 2 (CCR2), with CAS 445479-97-0 did not block the pro-fibrotic activity of Cytl1 in vitro. Collectively, our data suggest that Cytl1 plays an essential role in CF likely through activating the TGF-ß-SMAD signaling pathway. Although the receptor for Cyt1l remains to be identified, Cytl1 provides a novel platform for the development of anti-CF therapies.