Background & AimThe Phase I SECRET-HF trial (NCT05774509) assesses the effects of an investigational medicinal product (IMP), comprising the extracellular vesicle (EV)-enriched secretome of cardiovascular progenitor cells (CPC), on severely symptomatic patients with drug-refractory left ventricular (LV) dysfunction secondary to non-ischemic dilated cardiomyopathy (DCM).Methods, Results & Conclusion: MethodsThe IMP was prepared using current good manufacturing practices as follows. Human induced pluripotent stem cells (iPSC)-derived CPC were cultured to generate secretome-rich conditioned media (MC). The MC were clarified, and the EV were concentrated/isolated by tangential flow filtration. The IMP was extensively tested (ex. identity, purity, safety, and potency). The study was authorised by the French Regulatory Agency (Agence nationale de sécurité du médicament et des produits de santé), and approved by the Ethics Review Board of Île-de-France V. Approved inclusion / exclusion criteria were used to screen for eligible participants. Qualifying persons were included upon informed consent.Case ReportA first patient, with non-ischemic DCM due to a pathogenic variant of tropomyosin was included in the trial. At inclusion, he was in New York Heart Association (NYHA) class III, with LV end-diastolic and end-systolic volumes of 220 mL and 164 mL, respectively, and an ejection fraction of 25%. The participant received three secretome infusions of 20 x109 particles/kg each, three weeks apart (equivalent to the secretome of 106 mother cells/kg/infusion). All infusions were well tolerated without SAEs. Donor-specific antibody testing, repeated after each infusion and 28 days post-treatment, showed no allogeneic-immunization against the IMP. Encouragingly, the participant's levels of C-Reactive Protein decreased from 15.6 mg/L (at inclusion) to 1.9 mg/L (after three treatments). Also, eleven weeks after the first infusion, he had improved to NYHA Class II with stable echo parameters, and had a reduced need for diuretics (from 240 mg to 160 mg).DiscussionThis first-in-man case documents the safety of repeated administration of the secretome of iPSC-CPC as demonstrated by the lack of immune-inflammatory response or alloimmunization against the IMP. As the trial continues to enrol patients, it will hopefully confirm this initial, encouraging safety result and help assess whether this acellular approach, more easily manageable in the clinic than whole cells, is therapeutically efficacious.

The introduction of Chimeric Antigen Receptor (CAR)-T cells in the clinics has been a real milestone in the management of some malignant haematological diseases of poor prognosis by enabling complete and sustained remissions. The principle is to use lymphocytes (currently autologous in most cases) and to genetically engineer them ex vivo to make them co-express an antibody specific for a tumour-associated antigen and an activation signal triggering the produc-tion of cytotoxic factors and therefore allowing to neutralize the tumour cell upon recognition of the relevant antigen. The successful outcomes of CAR-T cells have led to broaden their indications to solid tumours and to consider now their possible extension beyond oncology, particularly to the cardiology area. Namely, the identification in fibrosis, a major hallmark of late-stage cardiomyopathies and a predictive factor of their worsening, of a highly expres-sed protein (Fibroblast Activation Protein) has led to investigate the effects of CAR-T cells directed against this protein. The initial experimental results in a murine model of hyperten-sive cardiomyopathy validate the efficacy of this approach for ablating myocardial fibrosis and concomitantly improving cardiac function. It now becomes mandatory to confirm these encoura-ging data, to check for the absence of toxicity and to streamline the current ex vivo production process which is still too time-consuming, complex and costly. This might be possible by leve-raging the technology which has allowed the successful development of anti-Covid-19 vaccines and relies on lipid nanoparticles targeting T lymphocytes for in vivo delivery of the messenger RNA encoding the target protein.& COPY; 2022 l'Acad & PRIME;emie nationale de m & PRIME;edecine. Published by Elsevier Masson SAS. All rights reserved.

L’introduction des Chimeric Antigen Receptor (CAR)-T cells en clinique a représenté un véritable tournant dans la prise en charge de certaines hémopathies malignes de mauvais pronostic en permettant des rémissions complètes et durables. Le principe est d’utiliser des lymphocytes (à ce jour le plus souvent autologues) et de les modifier génétiquement ex vivo pour leur faire co-exprimer un anticorps spécifique d’un antigène tumoral et un signal d’activation déclenchant une production de molécules cytotoxiques et permettant ainsi de neutraliser la cellule maligne une fois cet antigène reconnu. L’efficacité des CAR-T cells explique que l’on cherche à élargir leurs indications aux tumeurs solides et fait maintenant envisager des applications extra-oncologiques, notamment dans le domaine cardiaque. En effet, l’identification dans la fibrose, composante majeure et aggravante des cardiopathies parvenues à un stade avancé, d’une protéine fortement sur-exprimée (la Fibroblast Activation Protein) a conduit à explorer les effets de CAR-T cells dirigés contre cette protéine. Les premiers résultats expérimentaux dans un modèle murin de cardiopathie hypertensive valident l’efficacité de cette stratégie pour diminuer la fibrose myocardique et améliorer en parallèle la fonction cardiaque. Il faut maintenant conforter ces résultats encourageants, vérifier l’absence de toxicité et envisager une simplification de la procédure de production ex vivo actuelle, encore trop longue, complexe et coûteuse. Cela pourrait être possible en capitalisant sur la technologie qui s’est révélée efficace pour développer des vaccins anti-Covid 19 et repose sur l’utilisation de nanoparticules lipidiques ciblant les lymphocytes T pour leur transférer in vivo l’ARN messager codant pour la protéine ciblée.

In response to myocardial infarction (MI), extracellular vesicles (EVs), including large (lEVs) and small (sEVs), are released within and from the heart to facilitate intercellular communication and maintain cardiac homeostasis by transporting cargo to recipient cells. We investigated how glucose intolerance influences the intracardiac EV release post-MI and their content. B6J mice were fed chow (CD) or high-fat diet (HFD) for 3 months. MI was induced by permanent coronary artery ligation. EVs were isolated from left ventricles and quantified by tunable resistive pulse sensing. EVs were characterised by flow cytometry. EV miRNA content was determined by RNAseq and qPCR. Using cardiomyocyte specific GFP+ mice, plasma lEVs were analysed by flow cytometry to determine if cardiomyocyte EVs (CMEVs) are circulating. Labelled hypoxic cardiomyocyte cell line (HL-1) lEVs were injected in HFD/CD mice post-MI to determine target cells. In CD mice, EV release was significantly increased 24 h post-MI compared to sham. HFD lEV levels were significantly higher compared to sham and CD mice post-MI with no difference in sEV release between sham and MI HFD mice. Intracardiac lEVs originate from cardiomyocyte and endothelial cells in response to MI and MI + HFD respectively. qPCR analyses identified miRNA candidates that were modulated by MI and HFD. Intracardiac GFP + lEV levels were lower in HFD than in CD mice whereas levels of circulating GFP + lEVs were higher. In vivo biodistribution studies revealed a preferential uptake of hypoxic HL-1 lEVs by splenic myeloid cells in HFD spleens versus CD post-MI. Our results show that glucose intolerance modulates intracardiac EV release post-MI and their miRNA cargo. Circulating CMEV levels as well as their uptake by splenic myeloid cells are increased. Further investigations will aim to decipher the impact of the intracardiac EV miRNA mediated transfer in the diabetic heart post-MI.

Background & Aim Introduction: Extracellular vesicles (EV) recapitulate most of the cardioprotective effects of stem cells but their rapid washout from the myocardium could lead to a subsequent loss of therapeutic efficacy. Objective To load an injectable and biocompatible hydrogel with EV to allow their controlled and sustained release in the myocardium. Methods, Results & Conclusion Material: EV collected from HUVEC and labeled with green calcein were loaded in a hyaluronic acid hydrogel already used in clinical practice (Healon GV, Abbott). The presence of EV in the polymer was studied by confocal microscopy 4h after their inclusion within the gel. The rheological properties of the EV-loaded hydrogel were characterized by flow and oscillation measurements. To evaluate the ability of the biomaterial to deliver EV over time, Transwell assays were developed and release kinetics were assessed for 10 days with Nanoparticle Tracking Analysis (NTA) and imaging of the released EV (n=3). In parallel, the swelling of the gel was measured over time. Finally, the feasibility of intramyocardial injection of the hydrogel was tested in rats. Results Confocal microscopy of the gel containing labeled EVs displayed the presence of several fluorescent and round-shaped particles similarly sized as EV and spreading homogeneously throughout the thickness of the polymer, implying the successful incorporation of EV in the gel. The viscosity of Healon-EVs gel decreased upon shear increase (shear-thinning behavior), which allowed its convenient injectability. After being sheared, it immediately recovered its viscosity at rest. This non-thixotropic property as well the viscoelastic characteristics of the gel should favor extended residence time in vivo after injection. Quantitative (NTA) and qualitative (Imagestream) analysis showed that the number of released EV increased gradually from 8% to 52% over 10 days whereas without the hydrogel 70% of EV were released in one day. It highlights the ability of the biomaterial to support a sustained release of incorporated vesicles. The hydrogel swelled by absorbing solvent the first day before the polymer chains gradually disentangled, which allowed for the progressive release of the EV. In vivo, multiple injections of the gel in the myocardium could be performed in a straightforward fashion. Conclusion EV can be loaded in an injectable and clinically usable hyaluronic acid gel which seems a suitable vehicle for their sustained release in the myocardium. Introduction: Extracellular vesicles (EV) recapitulate most of the cardioprotective effects of stem cells but their rapid washout from the myocardium could lead to a subsequent loss of therapeutic efficacy. To load an injectable and biocompatible hydrogel with EV to allow their controlled and sustained release in the myocardium. Material: EV collected from HUVEC and labeled with green calcein were loaded in a hyaluronic acid hydrogel already used in clinical practice (Healon GV, Abbott). The presence of EV in the polymer was studied by confocal microscopy 4h after their inclusion within the gel. The rheological properties of the EV-loaded hydrogel were characterized by flow and oscillation measurements. To evaluate the ability of the biomaterial to deliver EV over time, Transwell assays were developed and release kinetics were assessed for 10 days with Nanoparticle Tracking Analysis (NTA) and imaging of the released EV (n=3). In parallel, the swelling of the gel was measured over time. Finally, the feasibility of intramyocardial injection of the hydrogel was tested in rats.

Abstract Introduction Extracellular Vesicles (EV) seem to mediate the benefits of cell therapy for ischemic heart failure. Although their mechanism of action remains poorly understood, one hypothesis is that they might trigger the generation of new cardiomyocytes. The doubly transgenic fate-mapping MerCreMer/ZEG mice model was thus used to distinguish whether these putative new cardiomyocytes originated from the division of preexisting ones (GFP+, Troponin T [TnT+], EdU+) or differentiated from endogenous progenitors, in which case they would stain positive for TnT+/EdU+ but negative for GFP. Methods Myocardial infarction was induced in 35 MerCreMer/ZEG mice by permanent occlusion of the left anterior descending coronary artery. Three weeks later, the surviving mice (n=18) with a left ventricular ejection fraction (LVEF) ≤45% received transcutaneous echo-guided injections in the peri-infarct myocardium of either EV (from 1.4 million human iPS-derived cardiovascular progenitor cells; 10 billion particles, n=9) or PBS (n=9); osmotic pumps were implanted to deliver EdU for 7 days in order to track the proliferation of new and native cardiomyocytes. Four-6 weeks after treatment all mice were evaluated by echocardiography (n=9 per group) and MRI (7 in each group), and then sacrificed for histological assessment, blindly. Results Based on echocardiography (MRI data pending), EV improved LVEF by 16% relative to baseline while a decrease of 4% was observed in the PBS group (p=0.46). The number of new cardiomyocytes (TnT+/EdU+/GFP+) did not significantly differ between the EV-treated hearts and the controls, and averaged 0.54% of the total heart cell content in infarct, peri-infarct and remote areas. However, EV treatment better preserved preexisting GFP+/WGA+/TnT+ cardiomyocytes in the peri-infarct area as their number was greater by 5.15% compared to PBS (32 sections analyzed for each mouse). Compared to the PBS control group, EV delivery was also associated with a 2.5% decrease in fibrosis, a reduction of infarct size by 14.9%, and an increase in angiogenesis in the peri-infarct area (with a between-group absolute difference of 71 capillaries, on the basis of isolectin staining). Conclusions EV secreted by iPS-derived cardiovascular progenitors improve the function of chronically infarcted hearts. Preservation of the existing cardiomyocyte pool and limitation of adverse remodeling and scarred tissue, likely favored by increased neoangiogenesis, are the main mechanisms mediated by the EV, while fate mapping allowed to exclude the generation of new cardiomyocytes.

Human embryonic stem cells (hESC) appear to be candidates for use in cell-based heart repair because they can be driven in vitro towards a cardiomyogenic lineage. These cells are able to engraft into infarct areas, differentiate into cardiomyocytes and subsequently improve heart function, although it remains uncertain whether these effects are due to the direct force-generating of ESC-derived differentiated cardiomyocytes or to their paracrine effects harnessing endogenous repair mechanisms. From a clinical perspective, the key issue is safety because of the risk that the final cell yield after transplantation may remain contaminated by residual, still pluripotent cells that could cause tumors. Optimization of purification therefore remains a major technical objective. Besides this hESC-specific manufacturing-related issue, hESC also share with all other cells aimed at cardiac repair problems of delivery, sustained survival and immune responses, which need to be addressed if their therapeutic potential is to be fully exploited.

In ischemic mitral regurgitation (IMR), leaflet tethering is caused by post-MI LV and annular remodeling. Severing second-order mitral chordae significantly decreases tethering and MR. We tested whether undersized ring annuloplasty can improve chordal cutting efficacy by reducing annulus-related tethering. Posterolateral MI created chronic remodeling and MR in 28 sheep. At 3 months, sheep were randomized to sham surgery vs isolated annuloplasty undersized by 2 sizes vs isolated bileaflet chordal cutting vs at the combined therapy (n=7 each). At baseline, chronic MI (3 months) and sacrifice (6.6 months) we measured LV volumes and ejection fraction (EF), wall motion score index (WMSi), MR Regurgitation fraction (MRRF) and vena contracta (VC), Mitral annulus area (MAA) and posterior leaflet (PL) restriction angle (PL to MAA) by 2D and 3D echo. All groups were comparable at baseline and chronic MI, with mild- moderate MR (MRVC 4.6±1.0mm, MRRF 24±2.6%) and MA dilatation (p<0.01). At sacrifice, LV end-systolic volume (ESV) increased by 108% in controls vs 28% with ring + chordal cutting, less than with each intervention alone (p<0.01). Also, MR progressed to moderate-severe in controls but decreased to trace with ring + chordal cutting vs mild-moderate with ring alone and trace-mild with chordal cutting alone (MRVC 5.9±1.1mm in controls, 2.0±0.7 with ring, 1.0±0.9 with chordal cutting, 0.5±0.08 with both, p<0.01). Ring alone did not improve PL mobility, but chordal cutting did alone or with ring (PL restriction angle 54±5° vs. 45±2.3° with ring, p=NS). In multivariate analysis, LVESV and MAA most strongly predicted MR (r 2 =0.82, p<0.01). Comprehensive annular and subvalvular repair provides the most effective long-term reduction of both chronic ischemic MR and LV remodeling.

Objectif Une limite importante en chirurgie cardiaque pediatrique est le manque de materiaux de remplacement de la voie de sortie ventriculaire droite (RVOT), doues de croissance et evitant les reoperations. Objectif : restaurer une RVOT fonctionnelle autologue a l’aide d’un patch en polydioxanone (PDO) biodegradable valve, ensemence de cellules souches mesenchymateuses (CSMs) autologues, dans un modele ovin en croissance. Methodes Des patchs en PDO biodegradables valves ensemences de CSMs autologues marquees par quantum dots ont ete implantes en position transannulaire sur la RVOT de 6 agneaux (suivi : 8 mois). Des patchs non ensemences ( n = 2) ou en pericarde autologue ( n = 2) constituaient les groupes controle. Resultats Echocardiographie et IRM demontrent l’absence de stenose et d’anevrysme de la neo-RVOT et son potentiel de croissance. Histologie et immunohistochimie objectivent moins de fibrose et de calcifications (0,08 % ± 0,03 % Ca2+), une degradation complete du polymere, l’apparition d’un neo-tissu organise et endothelialise avec matrice extracellulaire de type natif. Conclusion Un patch en PDO valve transannulaire ensemence de CSMs autologues restaure une RVOT vivante et fonctionnelle, et pourrait ameliorer le pronostic des cardiopathies droites congenitales.