Oral bioavailability of a noncoding RNA drug, TY1, that acts on macrophages.

All approved RNA therapeutics require parenteral delivery. Here we demonstrate an orally bioavailable formulation wherein synthetic noncoding (nc) RNA, packaged into lipid nanoparticles, is loaded into casein-chitosan (C2) micelles. We used the C2 formulation to deliver TY1, a 24-nucleotide synthetic ncRNA which targets the DNA damage response pathway in macrophages. C2-formulated TY1 (TY1C2) efficiently packages and protects TY1 against degradative enzymes. In healthy mice, oral TY1C2 was well-tolerated and nontoxic. Oral TY1C2 exhibited disease-modifying bioactivity in 2 models of tissue injury: 1) rat myocardial infarction, where a single oral dose of TY1C2 was cardioprotective, on par with intravenously-delivered TY1; and 2) mouse acute lung injury, where a single dose of TY1C2 attenuated pulmonary inflammation. Mechanistic dissection revealed that TY1C2 is not absorbed into the systemic circulation but is, instead, taken up by intestinal macrophages, namely those of the lamina propria and Peyer's patches. This route of absorption may rationalize why an antisense oligonucleotide against Factor VII, which acts on hepatocytes, is not effective when administered in the C2 formulation. Thus, some (but not all) ncRNA drugs are bioavailable when delivered by mouth. Oral RNA delivery and uptake, relying on uptake via the gastrointestinal immune system, has broad-ranging therapeutic implications.

A novel micropeptide, Slitharin, exerts cardioprotective effects in myocardial infarction.

PURPOSE: Micropeptides are an emerging class of proteins that play critical roles in cell signaling. Here, we describe the discovery of a novel micropeptide, dubbed slitharin (Slt), in conditioned media from Cardiosphere-derived cells (CDCs), a therapeutic cardiac stromal cell type. EXPERIMENTAL DESIGN: We performed mass spectrometry of peptide-enriched fractions from the conditioned media of CDCs and a therapeutically inert cell type (human dermal fibrobasts). We then evaluated the therapeutic capacity of the candidate peptide using an in vitro model of cardiomyocyte injury and a rat model of myocardial infarction. RESULTS: We identified a novel 24-amino acid micropeptide (dubbed Slitharin [Slt]) with a non-canonical leucine start codon, arising from long intergenic non-coding (LINC) RNA 2099. Neonatal rat ventricular myocytes (NRVMs) exposed to Slt were protected from hypoxic injury in vitro compared to a vehicle or scrambled control. Transcriptomic analysis of cardiomyocytes exposed to Slt reveals cytoprotective capacity, putatively through regulation of stress-induced MAPK-ERK. Slt also exerted cardioprotective effects in rats with myocardial infarction as shown by reduced infarct size 48 h post-injury. Conclusions and clinical relavance: Thus, Slt is a non-coding RNA-derived micropeptide, identified in the extracellular space, with a potential cardioprotective function.

Injury minimization after myocardial infarction: focus on extracellular vesicles.

Despite improvements in clinical outcomes following acute myocardial infarction, mortality remains high, especially in patients with severely reduced left ventricular ejection fraction (LVEF <30%), emphasizing the need for effective cardioprotective strategies adjunctive to recanalization. Traditional cell therapy has shown equivocal success, shifting the focus to innovative cardioactive biologicals and cell mimetic therapies, particularly extracellular vesicles (EVs). EVs, as carriers of non-coding RNAs and other essential biomolecules, influence neighbouring and remote cell function in a paracrine manner. Compared to cell therapy, EVs possess several clinically advantageous traits, including stability, ease of storage (enabling off-the-shelf clinical readiness), and decreased immunogenicity. Allogeneic EVs from mesenchymal and/or cardiac stromal progenitor cells demonstrate safety and potential efficacy in preclinical settings. This review delves into the translational potential of EV-based therapeutic approaches, specifically highlighting findings from large-animal studies, and offers a synopsis of ongoing early-stage clinical trials in this domain.

The ALPHA phase 1 study: pulmonary ArteriaL hypertension treated with CardiosPHere-Derived allogeneic stem cells.

BACKGROUND: Pulmonary Arterial Hypertension (PAH) is a progressive condition with no cure. Even with pharmacologic advances, survival remains poor. Lung pathology on PAH therapies still shows impressive occlusive arteriolar remodelling and plexiform lesions. Cardiosphere-derived cells (CDCs) are heart-derived progenitor cells exhibiting anti-inflammatory and immunomodulatory effects, are anti -fibrotic, anti-oxidative and anti-apoptotic to potentially impact several aspects of PAH pathobiology. In preclinical trials CDCs reduced right ventricular (RV) systolic pressure, RV hypertrophy, pulmonary arteriolar wall thickness and inflammation. METHODS: The ALPHA study was a Phase 1a/b study in which CDCs were infused into patients with Idiopathic (I)PAH, Heritable (H) HPAH, PAH-connective tissue disease (CTD) and PAH-human immunodeficiency virus (HIV). The study was IRB approved and DSMB monitored. Phase 1a, was an open label study (n = 6). Phase 1b was a double-blind placebo-controlled study (n = 20) in which half received 100 million CDCs (the maximum feasible dose from manufacturing perspective) and half placebo (PLAC) infusions. Right heart catheterization (RHC) and cardiac MR imaging (cMR) were performed at baseline and at 4 months post infusion. Patients were followed over a year. FINDINGS: No short-term clinical safety adverse events (AE) were related to the IP, the primary outcome measure. There were no adverse hemodynamic, gas exchange, rhythm or other clinical events following infusion and in the 1st 23 h monitored in hospital. There were no long-term AEs over 12 months noted, including unrelated limited hospitalizations. No immunologic short or long-term AEs were noted. We examined exploratory outcomes across multiple domains to determine encouraging signals to motivate future advanced phase testing. Phase 1a data showed encouraging observations for both 50 and 100 million CDC doses. Several encouraging findings favouring CDCs (n = 16) compared to placebo (n = 10) were noted. On cMR, the RV end diastolic volume (RVEDV) and index (RVEDVI) decreased with CDCs with a rise in the PLAC group. The 6-min walk distance was increased 2 months post infusion in the CDC group compared with PLAC. With PLAC, diffusing capacity (DLCO) decreased at 4 months but was unchanged with CDCs. Serum creatinine decreased with CDCs at 4 months. Encouraging observations favouring CDCs were also noted for RV fractional area change on echo and RV ejection fraction (RVEF) on cMR at 4 months. No differences were observed for mean pulmonary artery pressures or pulmonary vascular resistance. Review of long-term data to 12 months showed continued decline in DLCO for the PLAC cohort at 6 months with no change through 12 months. By contrast, CDC subjects showed an unchanged DLCO over 12-months. For parameters exhibiting early encouraging exploratory findings in CDC subjects, no further improvement was noted in long-term follow up through 12 months. INTERPRETATION: Intravenous CDCs were safe in both the short and long term in PAH subjects and thus may be safe in larger cohorts, in line with our extensive track record of safety in clinical trials for other conditions. Further, CDCs exhibited encouraging exploratory findings across several domains. Repeat dosing (quarterly, over one year) of intravenous CDCs has been reported to yield highly significant sustained disease-modifying bioactivity in subjects with advanced Duchenne muscular dystrophy. Because only single CDC doses were used here, the findings represent a lower limit estimate of CDC's potential in PAH. Upcoming phase 2 studies would logically use a repeat dosing paradigm. FUNDING: California Institute for Regenerative Medicine (CIRM). Project Number: CLIN2-09444.

Peptidyl arginine deiminase inhibition alleviates angiotensin II-induced fibrosis.

OBJECTIVES: The conversion of protein arginine residues to citrulline by calcium-dependent peptidyl arginine deiminases (PADs) has been implicated in the pathogenesis of several diseases, indicating that PADs are therapeutic targets. A recent study indicated that PAD4 regulates age-related organ fibrosis and dysfunction; however, the specific role of this PAD and its citrullination substrate remains unclear. We investigated whether pharmacological inhibition of PAD activity could affect the progression of fibrosis and restore heart function. METHODS: Cardiac hypertrophy was induced by chronic infusion of angiotensin (Ang) II. After 2 weeks of AngII infusion, a PAD inhibitor (Cl-amidine hydrochloride) or vehicle (saline) was injected every other day for the next 14 days together with the continued administration of AngII for a total of up to 28 days. Cardiac fibrosis and remodeling were evaluated by quantitative heart tissue histology, echocardiography, and mass spectrometry. RESULTS: A reverse AngII-induced effect was observed in PAD inhibitor-treated mice (n=6) compared with AngII vehicle-treated mice, as indicated by a significant reduction in the heart/body ratio (AngII: 6.51±0.8 mg/g vs. Cl-amidine: 5.27±0.6 mg/g), a reduction in fibrosis (AngII: 2.1-fold increased vs. Cl-amidine: 1.8-fold increased), and a reduction in left ventricular posterior wall diastole (LWVPd) (AngII: 1.1±0.04 vs. Cl-amidine: 0.78±0.02 mm). Label-free quantitative proteomics analysis of heart tissue indicated that proteins involved in fibrosis (e.g., periostin), cytoskeleton organization (e.g., transgelin), and remodeling (e.g., myosin light chain, carbonic anhydrase) were normalized by Cl-amidine treatment. CONCLUSION: Our findings demonstrate that pharmacological inhibition of PAD may be an effective strategy to attenuate cardiac fibrosis.

Three-vessel coronary infusion of cardiosphere-derived cells for the treatment of heart failure with preserved ejection fraction in a pre-clinical pig model.

Heart failure with preserved ejection fraction (HFpEF) is a major public health concern. Its outcome is poor and, as of today, barely any treatments have been able to decrease its morbidity or mortality. Cardiosphere-derived cells (CDCs) are heart cell products with anti-fibrotic, anti-inflammatory and angiogenic properties. Here, we tested the efficacy of CDCs in improving left ventricular (LV) structure and function in pigs with HFpEF. Fourteen chronically instrumented pigs received continuous angiotensin II infusion for 5 weeks. LV function was investigated through hemodynamic measurements and echocardiography at baseline, after 3 weeks of angiotensin II infusion before three-vessel intra-coronary CDC (n = 6) or placebo (n = 8) administration and 2 weeks after treatment (i.e., at completion of the protocol). As expected, arterial pressure was significantly and similarly increased in both groups. This was accompanied by LV hypertrophy that was not affected by CDCs. LV systolic function remained similarly preserved during the whole protocol in both groups. In contrast, LV diastolic function was impaired (increases in Tau, LV end-diastolic pressure as well as E/A, E/E'septal and E/E'lateral ratios) but CDC treatment significantly improved all of these parameters. The beneficial effect of CDCs on LV diastolic function was not explained by reduced LV hypertrophy or increased arteriolar density; however, interstitial fibrosis was markedly reduced. Three-vessel intra-coronary administration of CDCs improves LV diastolic function and reduces LV fibrosis in this hypertensive model of HFpEF.

Rejuvenating effects of young extracellular vesicles in aged rats and in cellular models of human senescence.

Rejuvenation of an old organism was achieved in heterochronic parabiosis experiments, implicating different soluble factors in this effect. Extracellular vesicles (EVs) are the secretory effectors of many cells, including cardiosphere-derived cells (CDCs) with demonstrated anti-senescent effect. 1. To determine the role of EVs (versus other blood fractions) on the rejuvenating effect of the young blood. 2. To evaluate the anti-aging properties of therapeutically administered EVs secreted by young-CDCs in an old organism. Neonatal blood fractioned in 4 components (whole blood, serum, EV-depleted serum and purified EVs) was used to treat old human cardiac stromal cells (CSPCs). CDCs were generated from neonatal rat hearts and the secreted CDC-EVs were purified. CDC-EVs were then tested in naturally-aged rats, using monthly injections over 4-months period. For validation in human samples, pediatric CDC-EVs were tested in aged human CSPCs and progeric fibroblasts. While the purified EVs reproduced the rejuvenating effects of the whole blood, CSPCs treated with EV-depleted serum exhibited the highest degree of senescence. Treatment with young CDC-EVs induce structural and functional improvements in the heart, lungs, skeletal muscle, and kidneys of old rats, while favorably modulating glucose metabolism and anti-senescence pathways. Lifespan was prolonged. EVs secreted by young CDCs exert broad-ranging anti-aging effects in aged rodents and in cellular models of human senescence. Our work not only identifies CDC-EVs as possible therapeutic candidates for a wide range of age-related pathologies, but also raises the question of whether EVs function as endogenous modulators of senescence.

Newt A1 cell-derived extracellular vesicles promote mammalian nerve growth.

Newts have the extraordinary ability to fully regenerate lost or damaged cardiac, neural and retinal tissues, and even amputated limbs. In contrast, mammals lack these broad regenerative capabilities. While the molecular basis of newts' regenerative ability is the subject of active study, the underlying paracrine signaling factors involved remain largely uncharacterized. Extracellular vesicles (EVs) play an important role in cell-to-cell communication via EV cargo-mediated regulation of gene expression patterns within the recipient cells. Here, we report that newt myogenic precursor (A1) cells secrete EVs (A1EVs) that contain messenger RNAs associated with early embryonic development, neuronal differentiation, and cell survival. Exposure of rat primary superior cervical ganglion (SCG) neurons to A1EVs increased neurite outgrowth, facilitated by increases in mitochondrial respiration. Canonical pathway analysis pinpointed activation of NGF/ERK5 signaling in SCG neurons exposed to A1EV, which was validated experimentally. Thus, newt EVs drive neurite growth and complexity in mammalian primary neurons.

High-Throughput Single-Cell Proteomic Analysis of Organ-Derived Heterogeneous Cell Populations by Nanoflow Dual-Trap Single-Column Liquid Chromatography.

Identification and proteomic characterization of rare cell types within complex organ-derived cell mixtures is best accomplished by label-free quantitative mass spectrometry. High throughput is required to rapidly survey hundreds to thousands of individual cells to adequately represent rare populations. Here we present parallelized nanoflow dual-trap single-column liquid chromatography (nanoDTSC) operating at 15 min of total run time per cell with peptides quantified over 11.5 min using standard commercial components, thus offering an accessible and efficient LC solution to analyze 96 single cells per day. At this throughput, nanoDTSC quantified over 1000 proteins in individual cardiomyocytes and heterogeneous populations of single cells from the aorta.

Current challenges and future directions for engineering extracellular vesicles for heart, lung, blood and sleep diseases.

Extracellular vesicles (EVs) carry diverse bioactive components including nucleic acids, proteins, lipids and metabolites that play versatile roles in intercellular and interorgan communication. The capability to modulate their stability, tissue-specific targeting and cargo render EVs as promising nanotherapeutics for treating heart, lung, blood and sleep (HLBS) diseases. However, current limitations in large-scale manufacturing of therapeutic-grade EVs, and knowledge gaps in EV biogenesis and heterogeneity pose significant challenges in their clinical application as diagnostics or therapeutics for HLBS diseases. To address these challenges, a strategic workshop with multidisciplinary experts in EV biology and U.S. Food and Drug Administration (USFDA) officials was convened by the National Heart, Lung and Blood Institute. The presentations and discussions were focused on summarizing the current state of science and technology for engineering therapeutic EVs for HLBS diseases, identifying critical knowledge gaps and regulatory challenges and suggesting potential solutions to promulgate translation of therapeutic EVs to the clinic. Benchmarks to meet the critical quality attributes set by the USFDA for other cell-based therapeutics were discussed. Development of novel strategies and approaches for scaling-up EV production and the quality control/quality analysis (QC/QA) of EV-based therapeutics were recognized as the necessary milestones for future investigations.

High Throughput Single Cell Proteomic Analysis of Organ Derived Heterogeneous Cell Populations by Nanoflow Dual Trap Single Column Liquid Chromatography.

Identification and proteomic characterization of rare cell types within complex organ derived cell mixtures is best accomplished by label-free quantitative mass spectrometry. High throughput is required to rapidly survey hundreds to thousands of individual cells to adequately represent rare populations. Here we present parallelized nanoflow dual-trap single-column liquid chromatography (nanoDTSC) operating at 15 minutes of total run time per cell with peptides quantified over 11.5 minutes using standard commercial components, thus offering an accessible and efficient LC solution to analyze 96 single-cells per day. At this throughput, nanoDTSC quantified over 1,000 proteins in individual cardiomyocytes and heterogenous populations of single cells from aorta.

MicroRNA-dependent suppression of biological pacemaker activity induced by TBX18.

Chemically modified mRNA (CMmRNA) with selectively altered nucleotides are used to deliver transgenes, but translation efficiency is variable. We have transfected CMmRNA encoding human T-box transcription factor 18 (CMmTBX18) into heart cells or the left ventricle of rats with atrioventricular block. TBX18 protein expression from CMmTBX18 is weak and transient, but Acriflavine, an Argonaute 2 inhibitor, boosts TBX18 levels. Small RNA sequencing identified two upregulated microRNAs (miRs) in CMmTBX18-transfected cells. Co-administration of miR-1-3p and miR-1b antagomiRs with CMmTBX18 prolongs TBX18 expression in vitro and in vivo and is sufficient to generate electrical stimuli capable of pacing the heart. Different suppressive miRs likewise limit the expression of VEGF-A CMmRNA. Cells therefore resist translation of CMmRNA therapeutic transgenes by upregulating suppressive miRs. Blockade of suppressive miRs enhances CMmRNA expression of genes driving biological pacing or angiogenesis. Such counterstrategies constitute an approach to boost the efficacy and efficiency of CMmRNA therapies.

Cell therapy attenuates endothelial dysfunction in hypertensive rats with heart failure and preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is defined by increased left ventricular (LV) stiffness, impaired vascular compliance, and fibrosis. Although systemic inflammation, driven by comorbidities, has been proposed to play a key role, the precise pathogenesis remains elusive. To test the hypothesis that inflammation drives endothelial dysfunction in HFpEF, we used cardiosphere-derived cells (CDCs), which reduce inflammation and fibrosis, improving function, structure, and survival in HFpEF rats. Dahl salt-sensitive rats fed a high-salt diet developed HFpEF, as manifested by diastolic dysfunction, systemic inflammation, and accelerated mortality. Rats were randomly allocated to receive intracoronary infusion of CDCs or vehicle. Two weeks later, inflammation, oxidative stress, and endothelial function were analyzed. Single-cell RNA sequencing of heart tissue was used to assay transcriptomic changes. CDCs improved endothelial-dependent vasodilation while reducing oxidative stress and restoring endothelial nitric oxide synthase (eNOS) expression. RNA sequencing revealed CDC-induced attenuation of pathways underlying endothelial cell leukocyte binding and innate immunity. Exposure of endothelial cells to CDC-secreted extracellular vesicles in vitro reduced VCAM-1 protein expression and attenuated monocyte adhesion and transmigration. Cell therapy with CDCs corrects diastolic dysfunction, reduces oxidative stress, and restores vascular reactivity. These findings lend credence to the hypothesis that inflammatory changes of the vascular endothelium are important, if not central, to HFpEF pathogenesis.NEW & NOTEWORTHY We tested the concept that inflammation of endothelial cells is a major pathogenic factor in HFpEF. CDCs are heart-derived cell products with verified anti-inflammatory therapeutic properties. Infusion of CDCs reduced oxidative stress, restored eNOS abundance, lowered monocyte levels, and rescued the expression of multiple disease-associated genes, thereby restoring vascular reactivity. The salutary effects of CDCs support the hypothesis that inflammation of endothelial cells is a proximate driver of HFpEF.

Repeated intravenous cardiosphere-derived cell therapy in late-stage Duchenne muscular dystrophy (HOPE-2): a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial.

BACKGROUND: Cardiosphere-derived cells (CDCs) ameliorate skeletal and cardiac muscle deterioration in experimental models of Duchenne muscular dystrophy. The HOPE-2 trial examined the safety and efficacy of sequential intravenous infusions of human allogeneic CDCs in late-stage Duchenne muscular dystrophy. METHODS: In this multicentre, randomised, double-blind, placebo-controlled, phase 2 trial, patients with Duchenne muscular dystrophy, aged 10 years or older with moderate upper limb impairment, were enrolled at seven centres in the USA. Patients were randomly assigned (1:1) using stratified permuted blocks to receive CAP-1002 (1·5 × 108 CDCs) or placebo intravenously every 3 months for a total of four infusions. Clinicians, caregivers, patients, and clinical operations personnel were fully masked to treatment groups. The primary outcome was the change in mid-level elbow Performance of Upper Limb version 1.2 (PUL 1.2) score at 12 months, assessed in the intention-to-treat population. Safety was assessed in all individuals who received an investigational product. This trial is registered with ClinicalTrials.gov, NCT03406780. FINDINGS: Between March 1, 2018, and March 31, 2020, 26 male patients with Duchenne muscular dystrophy were enrolled, of whom eight were randomly assigned to the CAP-1002 group and 12 to the placebo group (six were not randomised due to screening failure). In patients who had a post-treatment PUL 1.2 assessment (eight in the CAP-1002 group and 11 in the placebo group), the mean 12-month change from baseline in mid-level elbow PUL1.2 favoured CAP-1002 over placebo (percentile difference 36·2, 95% CI 12·7-59·7; difference of 2·6 points; p=0·014). Infusion-related hypersensitivity reactions without long-term sequelae were observed in three patients, with one patient discontinuing therapy due to a severe allergic reaction. No other major adverse reactions were noted, and no deaths occurred. INTERPRETATION: CAP-1002 cell therapy appears to be safe and effective in reducing deterioration of upper limb function in patients with late-stage Duchenne muscular dystrophy. Various measures of cardiac function and structure were also improved in the CAP-1002 group compared with the placebo group. Longer-term extension studies are needed to confirm the therapeutic durability and safety of CAP-1002 beyond 12 months for the treatment of skeletal myopathy and cardiomyopathy in Duchenne muscular dystrophy. FUNDING: Capricor Therapeutics.

Biological substrate modification suppresses ventricular arrhythmias in a porcine model of chronic ischaemic cardiomyopathy.

AIMS: Cardiomyopathy patients are prone to ventricular arrhythmias (VA) and sudden cardiac death. Current therapies to prevent VA include radiofrequency ablation to destroy slowly conducting pathways of viable myocardium which support re-entry. Here, we tested the reverse concept, namely that boosting local tissue viability in zones of slow conduction might eliminate slow conduction and suppress VA in ischaemic cardiomyopathy. METHODS AND RESULTS: Exosomes are extracellular vesicles laden with bioactive cargo. Exosomes secreted by cardiosphere-derived cells (CDCEXO) reduce scar and improve heart function after intramyocardial delivery. In a VA-prone porcine model of ischaemic cardiomyopathy, we injected CDCEXO or vehicle into zones of delayed conduction defined by electroanatomic mapping. Up to 1-month post-injection, CDCEXO, but not the vehicle, decreased myocardial scar, suppressed slowly conducting electrical pathways, and inhibited VA induction by programmed electrical stimulation. In silico reconstruction of electrical activity based on magnetic resonance images accurately reproduced the suppression of VA inducibility by CDCEXO. Strong anti-fibrotic effects of CDCEXO, evident histologically and by proteomic analysis from pig hearts, were confirmed in a co-culture assay of cardiomyocytes and fibroblasts. CONCLUSION: Biological substrate modification by exosome injection may be worth developing as a non-destructive alternative to conventional ablation for the prevention of recurrent ventricular tachyarrhythmias.

Biodistribution of unmodified cardiosphere-derived cell extracellular vesicles using single RNA tracing.

Extracellular vesicles (EVs) are potent signalling mediators. Although interest in EV translation is ever-increasing, development efforts are hampered by the inability to reliably assess the uptake of EVs and their RNA cargo. Here, we establish a novel qPCR-based method for the detection of unmodified EVS using an RNA Tracer (DUST). In this proof-of-concept study we use a human-specific Y RNA-derived small RNA (YsRNA) we dub "NT4" that is enriched in cardiosphere-derived cell small EVs (CDC-sEVs). The assay is robust, sensitive, and reproducible. Intravenously administered CDC-sEVs accumulated primarily in the heart on a per mg basis. Cardiac injury enhanced EV uptake in the heart, liver, and brain. Inhibition of EV docking by heparin suppressed uptake variably, while inhibition of endocytosis attenuated uptake in all organs. In vitro, EVs were uptaken more efficiently by macrophages, endothelial cells, and cardiac fibroblasts compared to cardiomyocytes. These findings demonstrate the utility of DUST to assess uptake of EVs in vivo and in vitro.