Cardiac regenerative therapy

Abstract

The present disclosure is directed to cardiac regenerative therapy to reduce the development of chronic heart failure in subjects selected from those having severe post-acute myocardial infarction (AMI), wherein said cardiac regenerative therapy comprises transendocardial injection of an efficient amount of said population of CD34+ cells in the left ventricular wall of said selected subjects.

Description

[0001] CARDIAC REGENERATIVE THERAPY

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to cardiac regenerative therapy based on the use of CD34+ cells, in particular autologous expanded CD34+ cells

[0004] BACKGROUND

[0005] The prevention of hospitalization and mortality associated with progression to heart failure following acute myocardial infarction (AMI) remains a major public health issue.1 2In the era of primary percutaneous coronary intervention, the rate of heart failure for hospitalization and death dropped to about 4% per year in AMI patients with left ventricular (LV) dysfunction,3-5but the event rate remains as high as 15% per year in patients presenting a large AMI, characterized by a decreased LV ejection fraction (LVEF), elevated cardiac biomarkers and microvascular obstruction (MVO)6despite optimal standard of care with betablockers, angiotensin-converting enzyme (ACE) inhibitors / angiotensin receptor / neprilysin (ARNI) inhibitors, sodium-glucose cotransporter 2 (SGLT2) inhibitors, and mineralocorticoid receptor antagonists (MRAs).4’57Over the past decades, therapy based on the myocardial injection of bone marrow cells (BMC) failed to translate into a promising strategy despite the capacity of those cells to improve ventricular remodelling and left ventricular (LV) function.89Due to their paracrine role stimulating angiogenesis and opposing remodelling,10’11CD34+ cells have been identified as the most efficient BMC cells for limiting the infarct size in patients with AMI.1213

[0006] Benefits of cell therapy post-AMI were found to be dependent on early treatment after AMI, the severity of LV dysfunction and the presence of MVO,8’1415as well as on the number of injected cells,16’17and the route of injection. Regarding the latter, intracoronary administration is associated with a lower retention of cells in the targeted tissue when compared to transendocardial injection.18However, despite some progress in the field, there is still a need to identify treatments for subjects with severe post-AMI, especially those subjects with persistent high-risk of subsequent adverse remodelling.

[0007] The applicant conducted a phase l / llb trial (NCT02669810) set up to assess the feasibility and efficacy trends of transendocardial injections of expanded autologous CD34+ cells in patients presenting with a severe AMI, in particular subjects characterized by(i) akinetic or dyskinetic left ventricular segments and Left Ventricular Ejection Fraction (LVEF) below 45% at day 2 (±2),

[0008] (ii) Left Ventricular Ejection Fraction (LVEF) remaining below 45% at day +8 (±3), (iii) microvascular obstruction (MVO) at day +8 (±3),

[0009] (iv) non-viable segment characterized by transmural extent of the infarct above 50% at day +8 (±3), and / or,

[0010] (v) a troponin peak with at least one value 70 times above the upper reference limit between day 0 and day 8,

[0011] day 0 being the day of last percutaneous coronary intervention or day of hospitalization if no stent has been implanted.

[0012] The results show for the first time favourable changes in remodelling in such selected population with high-risk of developing chronic heart failure.

[0013] SUMMARY

[0014] The present disclosure relates to a population of CD34+ cells, for use in a cardiac regenerative therapy to reduce the development of chronic heart failure in severe post-acute myocardial infarction (AMI) subject, wherein said cardiac regenerative therapy comprises transendocardial injection of an efficient amount of said population of CD34+ cells in the left ventricular wall, wherein said subject is selected among the subjects with severe AMI characterized by

[0015] (i) akinetic or dyskinetic left ventricular segments and Left Ventricular Ejection Fraction (LVEF) below 45% at day 2 (±2)

[0016] (ii) Left Ventricular Ejection Fraction (LVEF) remaining below 45% at day +8 (±3), (iii) microvascular obstruction (MVO) at day +8 (±3),

[0017] (iv) non-viable segment characterized by transmural extent of the infarct above 50%, at day +8 (±3), and / or,

[0018] (v) a troponin peak with at least one value 70 times above the upper reference limit between day 0 and day 8,

[0019] day 0 being the day of last percutaneous coronary intervention or day of hospitalization if no stent has been implanted.In specific embodiments, said subject is selected among the subjects with severe AMI characterized by

[0020] (i) akinetic or dyskinetic left ventricular segments and Left Ventricular Ejection Fraction (LVEF) below 45% at day 2 (±2)

[0021] (ii) Left Ventricular Ejection Fraction (LVEF) below 45% at day +8 (±3),

[0022] (iii) microvascular obstruction (MVO) at day +8 (±3),

[0023] (iv) above 50% transmural extent of the infarct, or akinetic or dyskinetic segments, at day +8 (±3), and,

[0024] (v) a troponin peak with at least one value 70 times above the upper reference limit between day 0 and day 8.

[0025] In specific embodiments, said population of CD34+ cells are administered within 3 months, for example within the second month, following the day of the AMI onset.

[0026] In specific embodiments, said CD34+ cells are expanded CD34+ cells, preferably autologous expanded CD34+ cells.

[0027] In specific embodiments, said population of CD34+ cells is obtained from peripheral blood mononuclear cells of said subject, after mobilization.

[0028] In specific embodiments, mobilization comprises daily administration of G-CSF for 5 days with a dose of 10|ig / kg / day before cell harvest, and, optionally, in combination with plerixafor on day 5 of administration of G-CSF.

[0029] In specific embodiments, a sample of 50 to 300 mL of whole blood is harvested the day after last G-CSF injection as the starting material for purifying and expanding CD34+ cells.

[0030] In specific embodiments, said population of CD34+ cells comprises at least 76% of viable CD34+ cells.

[0031] In specific embodiments, said population of CD34+ cells is a population of autologous expanded CD34+ cells obtained from a mobilized whole blood sample of 100 to 300 mL plus anticoagulant. In specific embodiments said CD34+ cells are obtained by(i) selecting CD34+ cells by immunoselection from a peripheral blood cell sample of said subject,

[0032] (ii) incubating said population of selected CD34+ cells and culturing in a culture medium for ex vivo expansion, and

[0033] (iii) selecting CD34+ cells from the ex vivo expanded cells.

[0034] In specific embodiments, the step of ex vivo expansion comprises culturing an initial amount of less than 13 x 106viable CD34+ cells for 5 to 15 days, for example about 9 days, in a CD34+ stem cell culture medium.

[0035] In specific embodiments, the quantity of VEGF in the cell culture supernatant at the end of the ex vivo expansion phase ranges from 100 pg / mL to 2800 pg / mL.

[0036] In specific embodiments, the expansion rate is comprised between 5 and 100, for example between 10 and 20.

[0037] In specific embodiments, said population of CD34+ cells injected in said subject comprises at least 6 x 106CD34+ cells.

[0038] In specific embodiments, said population of CD34+ cells comprises more than 76% CD34+ cells and more than 85% viable cells.

[0039] In specific embodiments, said subject receives said cardiac regenerative therapy in addition to standard of care for chronic heart failure, wherein said standard of care includes administering one or more of the following medicinal products:

[0040] Mineralocorticoid Receptor Antagonists,

[0041] Diuretics,

[0042] - Angiotensin Conversion Enzyme Inhibitors

[0043] - Angiotensin receptor blockers,

[0044] - Angiotensin receptor neprilysin inhibitor,

[0045] Sodium-Glucose Transport Protein 2 inhibitors,

[0046] Statins,

[0047] Platelet aggregation and

[0048] anticoagulants.In specific embodiments, said cardiac regenerative therapy promotes reverse remodelling of the left ventricle as assessed by a decrease in Left Ventricular Ejection Systolic Volume index (LVESVi) in the first 6 months post-treatment as compared to start of the treatment, for example a decrease of at least 10% at about 3 months as compared to start of treatment.

[0049] In specific embodiments, said cardiac regenerative therapy induces a decrease in N-terminal probrain natriuretic peptide type B (NT-proBNP) levels in the first 6 months post-treatment as compared to start of the treatment, for example a decrease of at least 25% of NT-pro-BNP secretion at about 3 months as compared to start of treatment.

[0050] In specific embodiments, said cardiac regenerative therapy reduces the infarcted zone as assessed by the number of transmural segments with late gadolinium enhancement >50%, or in particular >75%, in the first 6 months post-treatment, for example at least 1 , at least 2, at least 3, or at least 4.

[0051] BRIEF DESCRIPTION OF THE FIGURES

[0052] Figure 1 represents the treatment scheme of the clinical study.

[0053] Figure 2: Changes on cardiac magnetic resonance (CMR) remodelling parameters over 6 months. *One participant excluded from the analysis because of CMR contraindicated due to implantable cardioverter-defibrillator (ICD). **One participant excluded from the analysis for LVESVi and LVEDVi assessments because of missing body surface area data Adjusted means of change from baseline resulting from repeated measures ANCOVA analysis (with age and baseline value as covariates). LVESVi, left ventricular end systolic volume index; LVESV, left ventricle end systolic volume; LVEDVi, left ventricular end diastolic volume index; LVEDV, left ventricular end diastolic volume; LVEF, left ventricular ejection fraction; SE, standard error of the mean.

[0054] Figure 3: Changes on CMR Infarct size over 6 months.

[0055] Adjusted means of change from baseline resulting from repeated measures ANCOVA analysis (with age and baseline value as covariates). A. Number of segments with LGE >75%; B. HES in the territories diagnosed with MVO at baseline. Number of segments are assessed according to the 17 segments polar map. HES, hypersignal extension score; LGE, late gadolinium enhancement; MVO, microvascular obstructions; SE, standard error of the mean.* One participant excluded from the analysis because of CMR contraindicated due to implantable cardioverter device and two other participants excluded because of unexploitable LGE CMR. Figure 4: Change of NT-proBNP levels over 6 months

[0056] Adjusted means of ratio to baseline result from repeated measures ANCOVA analysis performed using log-transformed data. Graphical representation uses back transformed data. NT-proBNP, N-terminal pro-brain natriuretic peptide; S.E, standard error of the mean.

[0057] Nine participants (5 in the SoC group and 4 in the active group) were excluded because of the absence of baseline values and 11 participants (3 in the SoC group and 8 in the active group) were excluded because less than 4 values were available.

[0058] DETAILED DESCRIPTION

[0059] The present disclosure relates to a method of cardiac regenerative therapy, in particular for reducing the development of chronic heart failure in post-acute myocardial infarction (AMI) subject, wherein said method comprises transendocardial injection of an efficient amount of a population of CD34+ cells in the left ventricular wall of a subject in need thereof, wherein said subject is selected among the subjects with severe post-AMI characterized by

[0060] (i) akinetic or dyskinetic left ventricular segments and Left Ventricular Ejection Fraction (LVEF) below 45% at day 2 (±2),

[0061] (ii) Left Ventricular Ejection Fraction (LVEF) remaining below 45% at day +8 (±3), (iii) microvascular obstruction (MVO) at day +8 (±3),

[0062] (iv) non-viable segment characterized by transmural extent of the infarct above 50% at day +8 (±3), and / or,

[0063] (v) a troponin peak with at least one value 70 times above the upper reference limit between day 0 and day 8,

[0064] day 0 being the day of last percutaneous coronary intervention or day of hospitalization if no stent has been implanted.

[0065] General Definitions

[0066] The use of the articles “a”, “an”, and “the” in both the description and claims are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradictedby context. The terms “comprising”, “having”, “being of’, “including”, and “containing” are to be construed as open terms (i.e., meaning “including but not limited to”) unless otherwise noted. Additionally, whenever “comprising” or another open-ended term is used in an embodiment, it is to be understood that the same embodiment can be more narrowly claimed using the intermediate term “consisting essentially of’ or the closed term “consisting of’.

[0067] The term “about” or “ca.” has herein the meaning that the following value may vary for ± 20%, preferably ± 10%, more preferably ± 5%, even more preferably ± 2%, even more preferably ± 1%. The term "treating" or "treatment" or “therapy” as used herein comprises a treatment relieving, reducing or alleviating at least one symptom in a subject or effecting a delay of progression of a disease. For example, treatment can be the diminishment of one or several symptoms of a disorder or complete eradication of a disorder, such as favourable remodelling in post-AMI subjects, as suggested by changes in infarcted zone area, left ventricular dimensions and NT-proBNP assessments. Within the meaning of the present disclosure, the term "treat" also denotes to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and / or reduce the risk of developing or worsening a disease. As used herein in the context of the disclosed cardiac regenerative therapy, in one embodiment, the term “treatment” or “therapy” refers to significantly reducing the development of Chronic Heart Failure onset. Chronic heart failure may lead to cardiac transplantation, and chronic heart failure in high-risk patients is associated with high mortality, multiple complications, and poor quality of life. A reduction in the risk of chronic heart failure may be shown by a significant reduction of death, cardiac transplantation or frequency of heart failure hospitalization within about 12 months or 24 months from the date of treatment in a treated patient population with high-risk of heart failure as compared to a control high-risk population, for example receiving only the standard of care. The term “treatment” may also refer to promoting reverse cardiac remodelling, as determined for example by significant decrease of at least 25% of NT-pro-BNP secretion at about 3 months from start of treatment, or a decrease of at least 10% in Left Ventricle End Systolic Volume index (LVESVi) at about 6 months from start of treatment, or a reduction of infarcted zone as assessed by the number of transmural segments late with late gadolinium enhancement >50%, or in particular >75%, of at least 1, at least 2, at least 3 or at least 4 at about 6 months from start of treatment.

[0068] As used herein, the term “chronic heart failure” refers to a complex clinical syndrome that arises secondary to abnormalities of cardiac structure and / or function that impair the ability of the leftventricle to fill or eject blood.19It is a progressive condition where the heart muscle weakens and cannot pump blood efficiently.

[0069] The term "CD34+cells" as used herein refers to hematopoietic stem and progenitor cells and endothelial progenitor cells derived from human bone marrow that "are positive for" i.e. , "express", a stem cell antigen CD34.

[0070] As used herein, the phrase “therapeutically effective amount” or “efficient amount” in the context of a cardiac cell therapy refers to an amount of the population of cells, as the active principle, that will elicit a desired therapeutic response in at least a sub-population of subjects, for example, ameliorate the symptoms, alleviate conditions, slow or delay disease progression (such as chronic heart failure), or prevent a disease, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0071] The term "subject" or "patient" as used herein is intended to include animals, which are capable of suffering from or afflicted with a heart failure. Examples of subjects include mammals, e.g., humans, apes, monkeys, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In an embodiment, the subject is a human, e.g., a human suffering from, at risk of suffering from, or potentially capable of suffering from chronic heart failure.

[0072] The terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the cell therapy product, e.g. an efficient amount of the CD34+ cells as disclosed herein, and other therapeutic agent in a single subject in need thereof (e.g. a patient), and are intended to include treatment regimens in which the co-administered agents, i.e cell therapy product and the other therapeutic agent, are not necessarily administered by the same route of administration or at the same time.

[0073] The population of CD34+ cells for use in the cardiac regenerative therapy of the disclosure

[0074] The present disclosure relates to cardiac regenerative therapy involving administering, as the active principle, an efficient amount of a population of cells.

[0075] The term “population of cells” means that the population may contain different cell types. Typically, as used herein, the term “population of CD34+ cells” refers to a population of cells comprising at least 50% of the cells that express the CD34 protein on their surface. However, a population of CD34+ cells may also comprises in minor proportions CD34- cells. In specific embodiments, apopulation of CD34+ cells for use in the disclosed therapy comprises at least 50%, 60%, 70%, 80%, 90%, or more of CD34+ cells, for example as determined by flow cytometry methods. CD34 is a transmembrane phosphoglycoprotein that serves as a marker for hematopoietic stem cells and endothelial progenitor cells. These cells are characterized by their ability to differentiate into various cell types, including those involved in the formation of blood vessels and cardiac tissue. In the context of cardiac regenerative therapy, CD34+ cells are useful for their potential to promote angiogenesis, enhance myocardial repair, and improve cardiac function following ischemic injury. Total CD34+ cells represent approximately 0.5-1 % of total bone marrow derived mononuclear cells. CD34+ cells comprise hematopoietic stem / progenitor cells (HSPCs) as well as endothelial progenitor cells (EPCs). HSPCs can differentiate into all blood cell types and EPCs can differentiate into endothelial cells. CD34+ cells grow in suspension cultures.

[0076] In a specific embodiment, said population of CD34+ cells are a population of native cells obtained from mobilized peripheral blood mononuclear cells, after the administration of hematopoietic growth factor, such as G-CSF, optionally enriched in CD34+ cells for example using a separation step. As used herein, the term native means that the cells have not been expanded ex vivo. Methods for mobilizing CD34+ cells in a subject in need thereof are well-known in the art and for example described in20, which content is incorporated herein in its entirety by reference. In specific embodiment, mobilization comprises daily administration of G-CSF for about 5 days with a dose of about 10|ig / kg / day before cell harvest, and, optionally, in combination with plerixafor on day 5 of administration of G-CSF.

[0077] Examples of Granulocyte Colony-Stimulating Factor (G-CSF) include, without limitation:

[0078] 1. Filgrastim: A recombinant form of G-CSF used to stimulate the production of white blood cells, particularly neutrophils, in patients.

[0079] 2. Pegfilgrastim: A pegylated form of filgrastim, which has a longer half-life, allowing for less frequent dosing compared to filgrastim.

[0080] 3. Lenograstim: Another recombinant form of G-CSF, used similarly to filgrastim to boost white blood cell production in patients.

[0081] In a specific embodiment, said population of CD34+ cells are CD34+ cells obtained from a subject to be treated, i.e. autologous CD34+ cells.In other specific embodiment, CD34+ cells may be obtained from umbilical cord blood.

[0082] In other specific embodiment, CD34+ cells may be obtained from the bone marrow of a subject. In a specific embodiment, said population of CD34+ cells are expanded CD34+ cells. As used herein, “expanded cells” refers to cells which are obtained from a subject and cultured ex vivo for proliferation in an appropriate culture medium. In specific embodiments, the expansion rate is comprised between 5 and 100, for example between 10 and 30, and typically between 10 and 20. Additional steps of selection or purification for enrichment in CD34+ cells may be performed prior or after the expansion step. As used herein, the term “enrichment” refers to a step of either selecting or depriving certain subpopulation of cells in order to increase the relative proportion of a particular subpopulation in the population of cells that will be used in the therapy.

[0083] Hence, in a specific embodiment, said population of CD34+ cells are autologous expanded CD34+ cells, wherein the CD34+ cells are autologous cells obtained from mobilized peripheral blood mononuclear cells, after the administration of hematopoietic growth factor, such as Granulocyte Colony Stimulating Factor (G-CSF), in the subject to be treated, and further expanded ex vivo. In a specific embodiment, the population of CD34+ cells are autologous expanded CD34+ cells and they are obtained from an isolated population of autologous peripheral blood cells acquired early after an acute myocardial infarction, for example, within the first three months following the occurrence of an acute myocardial infarction, typically acquired 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 60 days or more after the occurrence of AMI. In a more specific embodiments, said isolated population of autologous peripheral blood cells is acquired within the second month following an acute myocardial infarction. In a more specific embodiment, the population of CD34+ cells are obtained by

[0084] (i) selecting or enriching CD34+ cells from a peripheral blood cell sample of said subject in need of cardiac regenerative therapy,

[0085] (ii) incubating said selected CD34+ cells and culturing in a culture medium for ex vivo expansion, and

[0086] (iii) selecting or enriching CD34+ cells from the ex vivo expanded cells.

[0087] For use in the above method, CD34+ cells may be enriched / selected by any techniques known to the skilled artisan. In certain embodiments, the CD34+ cells is enriched for cells expressing CD34cell antigen by fluorescence activated cell sorting (FACS). In some embodiments, isolation and / or purification of CD34+ cells is based on cell fractionation methods based on size and cell density, efflux of metabolic dyes, or resistance to cytotoxic agents.

[0088] In one embodiment, the CD34+ cells is enriched for CD34+ cells using a monoclonal anti-CD34 antibody and an immunomagnetic separation technique. Such immunomagnetic separation technique for selecting CD34+ cells includes for example immunomagnetic selection using CiiniMacs system (Miltenyi Biotec).

[0089] Methods for ex vivo expansion for CD34+ cells are well-known in the art and for example disclosed in21-23.

[0090] The CD34+ cells may be cultured for expansion for 5 to 12 days. In specific embodiments, the CD34+ cells may be cultured for expansion for 5, 6, 7, 8, 9, 10, 11, or 12 days. In a more specific embodiment, the CD34+ cells are cultured for expansion for 9 days.

[0091] Appropriate culture medium for ex vivo expansion include a culture medium comprising cytokines, for example, cytokines selected from interleukin 6 (IL6), interleukin 3 (IL3), Stem Cell Factor, Thrombopoietin, and Fms-Like Tyrosine kinase 3 Ligand.

[0092] In specific embodiments, the population of CD34+ cells for use in cardiac regenerative therapy is obtained by

[0093] (i) selecting CD34+ cells by immunoselection from total nucleated cells separated from the peripheral blood cell sample of said subject in need of cardiac regenerative therapy, (ii) incubating said selected CD34+ cells and culturing in a culture medium for ex vivo expansion, and

[0094] (iii) selecting CD34+ cells from the ex vivo expanded cells,

[0095] wherein the step of ex vivo expansion comprises incubating an amount of less than 13 x 106viable CD34+ cells for 9 days in a CD34+ stem cell culture medium.

[0096] In more specific embodiments which can be combined with the latter embodiment, the population of CD34+ cells is characterized in that the quantity of VEGF in the cell culture supernatant at the end of the ex vivo expansion phase ranges from 100 pg / mL to 2800 pg / mL, and for example from 200 pg / mL to 1400 pg / mL.An example of preferred autologous expanded CD34+ cells and methods to obtain such preferred cell therapy product for the method of use disclosed herein is detailed in the next section.

[0097] Preferred Cell Therapy Product for use in the cardiac regenerative therapy

[0098] The drug product in relation to cardiac regenerative therapy as disclosed herein will be referred hereafter as the Cell Therapy Product.

[0099] The Cell Therapy Product is a composition comprising, as the active substance, a suspension of an efficient amount of a population of CD34+ cells as described above.

[0100] In a preferred embodiment, the Cell Therapy Product is a composition comprising, as the active substance, a suspension of autologous expanded CD34+ cells.

[0101] Such suspension of autologous expanded CD34+ cells is formulated for injection in the subject, for example for intracoronary or transendocardial injection in the subject to be treated.

[0102] In specific embodiments, said suspension of autologous expanded CD34+ cells comprises at least 6 x 106CD34+ cells, for example from 6 x 106to 100 x 106CD34+ cells, more preferably between 6 x 106to 60 x 106CD34+ cells.

[0103] In specific embodiments, said suspension of autologous expanded CD34+ cells comprises more than 76% CD34+ cells, for example 76% to 100% and more than 85% viable cells, for example 85% to 100%.

[0104] The viability of CD34+ cells can be determined by FACS as described in24.

[0105] In specific embodiments, said suspension of autologous expanded CD34+ comprises 90 ± 10% of CD34+CD38+ cell subset.

[0106] In specific embodiments, said suspension of autologous expanded CD34+ cells are further characterized by VEGF secretion at the end of a nine days of an expansion phase, to be comprised between 100 pg / mL and 2800 pg / mL.

[0107] In specific embodiments, the suspension of cells may comprise additional excipients or carrier. For example, in a particular embodiment, said suspension of autologous expanded CD34+ cells is formulated with human albumin, for example 0.5% to 5% human albumin, and / or in a phosphate buffered saline (PBS) aqueous solution for trans-endocardial injection.A method for preparing a suspension of autologous expanded CD34+ cells comprises the following steps:

[0108] (i) Treating the subject with a colony stimulating factor, such as G-CSF agonist, for example Lenograstim for mobilizing CD34+ cells in vivo,

[0109] (ii) Harvesting a whole blood sample from the subject to be used as a starting material for manufacturing, typically a sample of 150 to 300 mL of whole blood,

[0110] (iii) Enriching said starting material in C34+ cells, for example by separation of total nucleated cells from said peripheral mobilized whole blood sample and immunoselection to obtain a population of cells enriched in CD34+ cells

[0111] (iv) Culturing said population of cells for CD34+ cell expansion, preferably for 5 to 12 days, for example 9 days,

[0112] (v) Enriching said population of expanded cells in CD34+ cells by immunoselection to obtain a population of expanded CD34+ cells

[0113] (vi) Optionally, formulating such population of expanded CD34+ cells for transendocardial injection in said subject with appropriate carrier, and / or excipients.

[0114] A specific method for preparing such suspension of autologous expanded CD34+ cells can be described as follows:

[0115] The subject to be treated is treated with a G-CSF agonist, for example Lenograstim, for 5 days before a whole blood harvest, typically, 220 ± 10 mL of whole blood, withdrawn by venipuncture. The whole blood harvest constitutes the starting material for the manufacturing process.

[0116] The first phase of the manufacturing process includes a first CD34+ cell purification by separation of total nucleated cells from said peripheral mobilized whole blood sample and immunoselection. Immunoselected CD34+ cells are then expanded, for example using the StemXpand® device as previously described24. Briefly, purified CD34+ cells are seeded into a dedicated culture bag containing the culture medium, e.g. StemFeed (CellProthera France) inside an incubator, for example the StemXpand® device and cultured, for 9 days at suitable conditions such as 37°C and 5% CO2.

[0117] After expansion, a second immunoselection is conducted and the resulting immunoselected CD34+ cells are resuspended, for example in phosphate buffered saline and human serum albumin, thus constituting the finished product.Detailed methods for preparing such suspension of autologous expanded CD34+ cells, in particular, the Protheracytes® cell therapy product (CellProthera) have also been described for example in24, or WO2024 / 105262 which content is incorporated in its entirety.

[0118] As used herein, the term “Protheracytes®” is the trade name for applicant’s human autologous expanded CD34+ cells for use in the cardiac regenerative therapy.

[0119] The StemXpand® is the trade name for an incubator system for the automated expansion of cells, such as CD34+ cells. It is described in US10,676,705B2.

[0120] The subject in need of the cardiac regenerative therapy

[0121] The present cell therapy is intended for a specific population of subjects at high-risk of developing chronic heart failure.

[0122] Indeed, the results of a phase l / llb study presented in the Examples show, for the first time, that a cardiac regenerative therapy is capable of reducing the risk of developing chronic heart failure in such high-risk population with a beneficial effect on infarct size and Left Ventricular remodelling. More specifically, the cardiac regenerative therapy disclosed herein is intended to be administered to subjects selected among the subjects with severe acute myocardial infarction (AMI), wherein said severe AMI is characterized by

[0123] (i) akinetic or dyskinetic left ventricular segments and Left Ventricular Ejection Fraction (LVEF) below 45% at day 2 (±2)

[0124] (ii) Left Ventricular Ejection Fraction (LVEF) remaining below 45% at day +8 (±3), (iii) microvascular obstruction (MVO) at day +8 (±3),

[0125] (iv) non-viable segment characterized by transmural extent of the infarct above 50%, at day +8 (±3), and / or,

[0126] (v) a troponin peak with at least one value 70 times above the upper reference limit between day 0 and day 8,

[0127] day 0 being the day of last percutaneous coronary intervention or day of hospitalization if no stent has been implanted.

[0128] As used herein, a subject with AMI is a subject which has been diagnosed of AMI according to the American Heart Association classification with infarct on left ventricular section.In particular, the subject to be treated will have LVEF below 45% and akinetic or dyskinetic segment at day 2 (±2), and LVEF remain below 45% at day +8 (±3), as determined for example using routine methods such as echography or cardiac magnetic resonance25.

[0129] More specifically, the subject to be treated is selected among the high risk patients with microvascular obstructions (MVO). The presence of a MVO segment can be identified and quantified at day +8 (±3) using cardiac magnetic resonance for example as described in626. In specific embodiments, the subject to be treated has above 50% transmural extent of the infarct at day +8 (±3) as determined for example by cardiac magnetic resonance (cMR), typically as described in27

[0130] In specific embodiments, said subject to be treated may typically have above 50% transmural extent of the infarct at day +8 (±3) and akinetic, or dyskinetic LV segments as assessed by cMR at day +8 (±3).

[0131] In specific embodiments, said subject is further characterized by having a troponin peak with at least one value 70 times above the upper reference limit between day 0 and day 8.

[0132] Hence, in a specific embodiment, said subject to be treated is selected among the subjects with severe acute myocardial infarction (AMI), wherein said severe AMI is characterized by the four features defined below:

[0133] (a) akinetic or dyskinetic left ventricular segments and Left Ventricular Ejection Fraction (LVEF) below 45% at day 2 (±2)

[0134] (b) Left Ventricular Ejection Fraction (LVEF) remaining below 45% at day +8 (±3),

[0135] (c) microvascular obstruction (MVO) at day +8 (±3),

[0136] (d) non-viable segment characterized by transmural extent of the infarct above 50%, at day +8 (±3), and / or,

[0137] (e) a troponin peak with at least one value 70 times above the upper reference limit between day 0 and day 8.

[0138] In a specific embodiment, the subject in need thereof is a revascularized myocardial infarction patient. The term “revascularized” as used in this embodiment refers to the successful placement of a stent.

[0139] The method of treatmentAs disclosed herein, the cardiac regenerative therapy comprises transendocardial injection of an efficient amount of said population of CD34+ cells in the left ventricular wall of a subject in need thereof, such as the high-risk population of patients as described in the previous section.

[0140] According to some embodiments, transendocardial injection includes one or more injections of said population of CD34+ cells in the peri-infarct border zone, for example, as described in28. Transendocardial administration of CD34+ cells preferably involves a precise and minimally invasive procedure to deliver the Cell Therapy Product directly into the myocardium. For example, the process begins with the patient undergoing a pre-injection evaluation, which may include cardiac magnetic resonance imaging (cMR) to assess scar thickness and confirm the absence of left ventricular thrombus. Once the patient is prepared, the procedure is performed using a catheter-based system, typically via the femoral artery. The catheter is advanced retrogradely into the left ventricular cavity under fluoroscopic guidance. A specialized injection catheter, such as the Helical™ Infusion Catheter, is used to ensure accurate delivery of the cells. The catheter is positioned at the peri-infarct border zone, and multiple injections, typically 10-15 injections of 0.5 to 1 mL each, are administered into the targeted myocardial tissue. This method enhances cell retention and promotes myocardial repair by delivering the cells directly to the site of injury. Following the procedure, the patient is monitored for any immediate complications, such as pericardial effusion, through echocardiography conducted immediately after the procedure and again six hours later. This approach aims to maximize the therapeutic potential of the CD34+ cells by ensuring their optimal localization and integration into the damaged cardiac tissue.

[0141] Hence in some embodiments, the disclosure provides a method for reducing the risk of heart failure, said method comprising

[0142] (i) Isolating CD34+ cells from bone marrow, peripheral blood or cord blood,

[0143] (ii) Expanding and selecting said CD34+ cells to obtain a population of expanded CD34+ cells,

[0144] (iii) Administering an efficient amount of said population of expanded CD34+ cells in said subject via transendocardial injection into the targeted myocardial tissue.

[0145] In specific embodiments, said population of expanded CD34+ cells are autologous expanded CD34+ cells as described in the previous sections.The population of CD34+ cells, typically autologous expanded CD34+ cells, can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.

[0146] Sterile injectable solutions can be prepared by incorporating the cells utilized in practicing the present cardiac regenerative therapy in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavouring agents, colours, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

[0147] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminium monostearate and gelatine. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the multipotent stem cells, endothelial progenitor cells, mesenchymal stem cells, mononuclear cells, or progenitors or progeny thereof.The compositions can be isotonic, i.e. , they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents, such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.

[0148] Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose is preferred because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form).

[0149] In some embodiments, said population of CD34+ cells is administered to said subject in need thereof within 6 months following the AMI onset, for example within 3 months, typically within 2 months following the AMI onset, and for example within the second month following the AMI onset. In specific embodiments, the cardiac regenerative therapy does not include any further injection of CD34+ cells following the first injection after the AMI.

[0150] The skilled person will determine the amount of CD34+ cells to be administered to the subject in need thereof.

[0151] In specific embodiments, an efficient amount of said population of CD34+ cells comprises 6 to 100 x 106CD34+ cells, and for example 6 to 60 x 106CD34+ cells.

[0152] In certain embodiments, said efficient amount is sufficient to promote reverse remodelling of the left ventricle as assessed by a decrease in Left Ventricular Ejection Systolic Volume index (LVESVi) in the first 3 or 6 months post-treatment, as compared to start of the treatment, for example a decrease of at least 10% at 3 months post-treatment as compared to start of treatment.In certain embodiments, said cardiac regenerative therapy induces a decrease in N-terminal probrain natriuretic peptide type B (NT-proBNP) levels in the first 3 or 6 months post-treatment as compared to start of the treatment, for example, a decrease of at least 25% of NT-pro-BNP secretion at about 3 months as compared to start of treatment.

[0153] In certain embodiments, said cardiac regenerative therapy reduces the infarcted zone as assessed by the number of transmural segments with late gadolinium enhancement >50%, or in particular >75%, scores in the first 3 or 6 months post-treatment as compared to start of the treatment, for example, a reduction of at least 1 , at least 2, at least 3, and preferably at least 4 segments of the infarcted zone at about 6 months as compared to start of treatment.

[0154] In specific embodiments, said population of CD34+ cells is co-administered on top of a standard of care therapy for treating acute myocardial infarction, for example, a therapy comprising administering an efficient amount of one or more of the following medicinal products:

[0155] Mineralocorticoid Receptor Antagonists,

[0156] Diuretics,

[0157] - Angiotensin Conversion Enzyme Inhibitors,

[0158] - Angiotensin receptor blockers,

[0159] - Angiotensin receptor neprilysin inhibitor,

[0160] Sodium-Glucose Transport Protein 2 inhibitors

[0161] Statins,

[0162] Platelet aggregation, and,

[0163] - Anticoagulants.

[0164] Hereinafter, further aspects of the cardiac regenerative therapy of the present disclosure are described in more details and specifically with reference to examples, which however are not intended to limit the present invention.

[0165] EXAMPLES

[0166] Example 1: A drug substance for use according to the present disclosure

[0167] Short description of the active substance

[0168] Autologous expanded CD34+ stem cells

[0169] Brief description of the finished productSuspension of expanded CD34+ Stem Cells conditioned in syringes for an autologous endocardiac injection.

[0170] After mobilisation, by administering G-CSF to the patient per the clinical protocol, CD34+ cells are collected from the patient via a whole blood withdrawal, the autologous non-expanded CD34+ cells are enriched, then expanded and purified after expansion to produce ProtheraCytes®. Structure of the drug substance

[0171] The drug substance consists of expanded autologous CD34+ cells. The cells are collected from peripheral blood after mobilisation from the blood marrow, with G-CSF (Lenograstim).

[0172] The mobilised whole blood is then processed to remove red blood cells through a sedimentation step (yield of 91.0±16.5%), followed by a CD34+ cell immunoselection step (yield of 79.7±16.7%) at Day 0 (DO). Cells are then put in culture in a 37°C, 5% CO2 monitored environment, and then enriched after expansion by immunoselection to obtain a CD34+ cell purity > 76%. Other cell phenotypes present in the drug substance can be:

[0173] CD 14+ (Monocytes)

[0174] - CD15+ (Granulocytes)

[0175] - CD 19 / 20+ (Lymphocytes B)

[0176] CD56+ (Lymphocytes NK)

[0177] - CD2 / 3+ (Lymphocytes T)

[0178] Growth and differentiation factors which are recombinant proteins (IL-3, IL-6, SCF, TPO and FLT-3 L) present in a custom formulated cytokine mix, regulate the survival and self-renewal of the stem cells along with proliferation and differentiation during the cell expansion.

[0179] The expanded CD34+ cells are formulated as a fresh suspension in 2% HSA / PBS for trans-endocardial injection.

[0180] Example 2: Methods of preparing expanded CD34+ cells for use in cardiac regenerative therapy

[0181] The method for preparing expanded CD34+ cells may comprise the following steps:

[0182] 1 / Patient G-CSF mobilization,

[0183] 2 / Sampling mobilized blood,3 / Isolating total nucleated cells from sampled mobilized blood,

[0184] 4 / Immunoselecting CD34+ cells from isolated total nucleated cells

[0185] 5 / Amplification of immunoselected CD34+ cells,

[0186] 6 / Immunoselecting CD34+ cells from amplified cells,

[0187] 7 / Harvesting immunoselected CD34+ cells and,

[0188] 8 / Formulating finished product for use in cardiac regenerative therapy.

[0189] Example 3: Results of Phase l / llb Study - a multicentre phase l / llb randomised controlled open-label with blinded evaluation trial.

[0190] Study design

[0191] The trial design of the study EXCELLENT was previously published.28

[0192] Participants were randomized 3:1 to receive standard of care (SoC) plus transendocardial injections of expanded autologous CD34+ cells (active group) or SoC alone as defined by current guideline-directed medical therapy for patients with AMI associated with LV dysfunction.2529Study population

[0193] Full inclusion and exclusion criteria have been previously detailed.28Briefly, eligible participants had to be > 18 and < 85 years-old, to present with AMI with or without ST segment elevation, an elevation of plasma cardiac troponin concentration >70 times the upper reference limit. Inclusion criteria were a persistent LVEF < 50%, and the identification of non-viable (transmural scar extent >50%) and akinetic, or dyskinetic LV segments assessed by cardiac magnetic resonance (CMR) performed 8 ± 3 days after AMI.

[0194] ProtheraCytes® manufacturing and injection procedure

[0195] Within 45 days after randomization, participants of the active group were treated with Lenograstim for 5 days prior to blood harvest. ProtheraCytes® (CellProthera, France) were manufactured by using an incubator, here specifically the StemXpand® industrialized platform as previously described24,28. Expansion rates and characterization of ProtheraCytes® in terms of purity andviability were determined as previously described.24The average number of CD34+ cells obtained after expansion was 20 x 106, corresponding to a 16-fold expansion rate (Table 1).

[0196] Table 1: Expansion rate and characteristics of CD34+ cells after expansion

[0197] <

[0198]

[0199] ProtheraCytes® were delivered the next day after expansion to the hospital’s central pharmacy and transferred to the catheterization laboratory for administration. Injections were performed in all participants randomized to the active group, except when quality control specifications were not met (in terms of quantity, viability and purity), in cases of patient’s choice or cardiological events occurring after randomization. Prior to the transendocardial injection, participants underwent CMR to measure wall thickness and confirm the absence of LV thrombus. LV transendocardial injections via the femoral route up to the LV cavity were performed by trained investigators by using the Helical™ Infusion (Helix™) Catheter in combination with the Universal Deflectable Guide (Morph®) Catheter (BioCardia, Sunnyvale CA, USA) as previously described.28,30Fifteen transendocardial injections (1 mL each) were performed by retrograde catheterization. Participants underwent a control echocardiography right after the procedure and 6 h later to check the absence of pericardial effusion.

[0200] Outcome measures

[0201] Follow-up assessments were performed at 1 , 3 and 6 months, and included physical examination, electrocardiogram (ECG), echocardiography, estimated glomerular filtration rate, blood cell count,natriuretic peptide, and quality of life with SF36 questionnaires. Assessments at 3 and 6 months also include CMR, an ECG and the assessment of the New York Heart Association (NYHA) functional stage. Single photon emission computed tomography (SPECT) could be optionally performed at baseline and 6 months, according to availability.

[0202] The primary endpoint was the 6-month incidence of major adverse cardiac events (MACEs) that were adjudicated and confirmed by an independent and blinded Clinical Events Committee (CEC). MACEs were defined as the composite incidence of all-cause death, heart failure-associated hospitalizations or urgent visits, non-fatal recurrent Ml, non-fatal strokes, or other cardiovascular hospitalizations. They also include procedure-associated events leading to hospitalization prolongation. The main secondary endpoints were changes in LVESVi and of the viability of infarcted segment(s) defined as late gadolinium enhancement (LGE) >75% from baseline to 6 months assessed by CMR. Other secondary endpoints included other markers such as CMR LVESV, LV End Diastolic Volume (LVEDV), LVEDV index (LVEDVi), and LVEF, and quality of life. The quality of life was assessed by using SF-36 questionnaires including 8 subdomains31: physical functioning, role physical, bodily pain, general health, vitality, social functioning, role emotional, and mental health.

[0203] The number of segments with or without MVO were identified and quantified as previously described.28The transmural scar extent was assessed by CMR by counting the number of segments using a 17 segments model . The summed perfusion defect scores were also assessed via optional perfusion 99mTc SPECT (when available)32in areas with signs of transmural infarction (absorption <50%). All CMRs and SPECTs were reviewed by a central blinded Corelab. In exploratory analyses,, the change in NT-ProBNP levels over the 6-month period was assessed, when available, as an exploratory endpoint. The change in the summed hypersignal extension score (HES)27in the cardiac segments diagnosed with MVO at baseline was also assessed. Statistical analysis

[0204] The sample size to be analysed was set to 33 subjects in the treatment arm and 11 subjects in the control arm. This sample size has been chosen considering that the expected proportion of MACE will be low (i.e., lower than 20%). This corresponds to a number of subjects experiencing MACE equal or lower than 6 among 33 subjects in the active group. The width of the confidenceinterval of the matching frequencies (0 / 33 to 6 / 33) ranges from 10.6 to 28.5%, which was deemed to be of sufficient accuracy.

[0205] All analyses were conducted on the per-protocol population defined as all participants randomized, and for the participants in the active group, those who had received at least one transendocardial injection of ProtheraCytes®. Efficacy analyses were conducted for all participants who had completed evaluable assessments and who did not have heart transplant or recurrent-Ml during the follow-up period. Intra-group 95% confidence intervals for measures at 3 or 6 months from baseline were calculated from repeated measures ANCOVA analyses with age and baseline values as a covariables. Descriptive results on the quality of life are presented as mean values and interquartileintervals.

[0206] Results

[0207] The trial was performed between January 2016 and September 2023, at 13 clinical sites in France and the UK. A total of 146 participants were considered eligible for the study, of whom 69 were not included mostly due to screen failure (mainly LV thrombus and recovering LVEF) (Figure 1). Seventy-seven participants were randomized but 28 participants of the active group did not receive ProtheraCytes®, mainly due to out of specifications and LV thrombi. Finally, 33 participants were considered for per protocol analysis. The mean time between AMI and lenograstim administration was 29 ± 11 days. The average number of CD34+ cells obtained after expansion was 20*106, corresponding to a 16-fold expansion rate (Table 1). In treated patients, purity and viability of CD34+ cells after expansion were found to be more than 90% (Table 1 ).

[0208] Participant characteristics

[0209] Participants had a mean age of 57 years, and 86% were male (Table 2). CMR-associated features were consistent with the inclusion criteria, with a mean LVEF lower than 40%, large infarct sizes, and high NT-proBNP levels. Overall, there were no substantial differences between the two groups at baseline, except for the mean participants age (55 years in the active group vs 62 years in the SoC group). Most participants had MVO at baseline in both groups extending in average over five cardiac segments. Participants received optimal medication, including antiplatelet agents, and medications for heart failure prevention.

[0210] Table 2: Baseline characteristics

[0211]

[0212] >

[0213] <

[0214]

[0215] * n=13 in the SoC group; ** n=29; n=10; # n=20; ## n=10; § n=32

[0216] ACE, angiotensin-converting enzyme; ARB, Angiotensin receptor blocker; NYHA, New York Heart Association; RAS, Renin-Angiotensin System; SGLT2, Sodium-glucose co-transporter 2Primary Endpoint

[0217] Eleven MACE were reported in 10 participants of the active group (10 / 33, 30.3 [95% confidence interval 15.6 to 48.7] %), which included one heart failure hospitalization, one Ml and nine transendocardial procedure related events. There were no death nor unexpected serious adverse event related to the expanded autologous CD34+ cells. Five MACE were reported in 3 participants in the SoC group (3 / 16, 18.8 [4.0 to 45.6] %). All were heart failure-related hospitalizations, one of whom underwent subsequent heart transplantation.

[0218] Main secondary outcomes

[0219] CMR analyses of LVESVi at 6 months were available in 14 (88%) and 30 (91%) participants in the SoC and active groups, respectively (Figure 2, Table 4). There were no demonstrable differences in LVESVi in the active (-5.7 [-12.1 to +0.7] mL / m2) and SoC (+0.2 [-9.3 to +9.6] mL / m2) groups.

[0220] Table 4: Changes in CMR remodelling endpoints and infarct size over 6 months Active group Standard of care group (n=30)* (n=15)**

[0221] Change from Baseline Change from Baseline Endpoint Adjusted means (95% Cl) Adjusted means (95% Cl)

[0222] at M3 at M6 at M3 at M6

[0223] -5.4 -5.7 _4 Q +0.2 LVESVi, mL / m2(-11.8; +1.0) (-12.1; +0.7) (-13.5; +5.4) (-9.3; +9.6)

[0224] -11.7 -11.8 -10.4 _03 LVESV, mL (-23.8; +0.3) (-23.9; +0.2) (-27.7; +6.9) (-17.6; +17.0)

[0225] -1.6 -3.2+48 +8.6 LVEDVi, mL / m2(-8.5; +5.4) (-10.1; +3.8) (-5.5; +15.1) (-1.7; +18.9)

[0226] -4.9 -7.5 +9.7 +174 LVEDV, mL (-18.3; +8.5) (-20.9; +5.9) (-9.5; +28.9) (-1.8; +36.7)

[0227]

[0228] Infarct size Active group (n=28)** SoC group (n=15)§

[0229] At 3 months At 6 months At 3 months At 6 months LGE > 75% -1-3 -1.7 -0.9 -1.0 segments, n '2 2: -°-4) '2 6:0 9) (-2.2; -0.3) (-2.2; +0.2)Active group Standard of care group (n=30)* (n=15)**

[0230] Change from Baseline Change from Baseline Endpoint Adjusted means (95% Cl) Adjusted means (95% Cl)

[0231] at M3 at M6 at M3 at M6

[0232] -2.1 -3.2

[0233]

[0234] HES, MVO area (-3.3; -0.9) (-4.3; -2.0) (-3.1 ; -0.2) (-3.5; -0.3)

[0235] *One participant excluded from the analysis because of CMR contraindicated due to implantable cardioverter device. **Two other patients excluded from the analysis because of unexploitable LGE CMR. § One participant excluded from the analysis for LVESVi and LVEDVi assessments because of missing body surface area. Adjusted means of change from baseline result from repeated measures ANCOVA analysis (with age and baseline value as covariates). Cl, confidence interval; HES, hypersignal extension score; LGE, late gadolinium enhancement; LVEDV, left ventricular end diastolic volume; LVEDVi, left ventricular end diastolic volume index; LVEF, left ventricular ejection fraction; LVESV, left ventricle end systolic volume; LVESVi, left ventricular end systolic volume index; MVO, microvascular obstructions.

[0236] CMR analyses of infarct sizes, were available in 15 (94%) and 28 (85%) participants in the SoC and active groups, respectively. The number of segments with transmural extend > 75% (LGE > 75%) appeared to be reduced in both active group (-1.7 [-2.6; -0.9]), and SoC group (-1.0 [-2.2 to +0.2]) (Figure 3. Table 4).

[0237] SPECT perfusion defect score were available and analysable in 9 (56%) and 17 (51 %) participants in the active and SoC groups respectively (Table 5). SPECT perfusion defects in the areas with signs of transmural infarction (absorption <50% at baseline) appeared to be reduced in the active (-1.7 [-2.9 to -0.6]) but not in SoC (-0.4 [-2.0 to +1.2]) groupsTable 5: Change in SPECT perfusion defect score over 6 months in area with sign of transmural infarct

[0238] Standard of Care group Active group N=9 N=17 Change from Baseline Change from Baseline Endpoint

[0239] Adjusted means (95% Cl) Adjusted means (95% Cl) Transmural area -0.4 -1.7 (Baseline absorption <50%) (-2.0; +1.2) (-2.9; -0.6)

[0240] Adjusted means of change from baseline are resulting from repeated measures ANCOVA analysis (with age and baseline value as covariates).

[0241] Exploratory outcomes

[0242] Serum NT-proBNP levels were available at baseline and follow up in 7 (37%) and 19 (58%) participants in the SoC and active groups, respectively (Figure 4). The ratios (±95 % Cl) at 1, 3 and 6 months from baseline were 0.48 [0.35-0.66], 0.30 [0.22; 0.41], 0.24 [0.17; 0.33], respectively, in the active group, and 0.65 [0.38; 1.12], 0.49 [0.29; 0.85] and 0.34 |0.20; 0.59], respectively, in the SoC group.

[0243] When considering segments with MVO at baseline, changes in the HES at 6 months were available and analysable in 28 (85%) and 15 (94%) of participants in the active and SoC groups, respectively (Table 5). A decrease from baseline was observed in both the active (-3.2 [-4.3 to -2.0]) and the SoC (-2.1 [-3.3 to -0.9]) groups.

[0244] Quality of life

[0245] Complete SF-36 questionnaires were available in 12 (75%) and 31 (94%) participants in the SoC and active groups, respectively. All 8 component scores were numerically increased over the study period in the active group and 4 components in the SoC group. In the active group a median (Q1 ; Q3) increase of +15% (5; 33) and +13% (0; 38) was observed for physical and social functioning scores respectively. Corresponding changes in the SoC group were +5 (-8; 20)% and 0 (-6; 38)%. (Table 6).Table 6: Changes in the SF-36 components over 6 months

[0246]

[0247] *Three participants (two in the SoC group and one in the active group) excluded from the analysis due to the absence of baseline value, and three other participants (two in the SoC group and one in the active group) excluded because the number of available values was less than 4. SD, standard deviation of the mean

[0248] Discussion

[0249] The above-described study is a prospective randomised open-label trial with blinded endpoint trial is the first to investigate the safety and feasibility of transendocardial injections of expanded autologous mobilized CD34+ cells in patients with a recent large myocardial infarction who are at risk of developing subsequent heart failure. We have shown that the treatment was feasible but was associated with procedural complications. Favourable signals were observed on LV remodelling and viability in the active group at 6 months, suggesting a benefit on left ventricle remodelling and progression of heart failure.

[0250] We successfully enriched our trial population to include patients who were at high risk of developing heart failure as demonstrated by the marked elevation in baseline troponin and NT- proBNP concentrations, and substantial LV dysfunction evidenced by the low baseline LVEF, transmurality and extent of akinesia or dyskinesia and the high rate of MVO. A high proportion of screen failures (33%) probably due to reversible myocardial stunning33and LVEF recovery with modern SoC therapy. This justifies the selection of a delayed (8 days) timing for screening CMR, targeting a population with persistent high risk of subsequent adverse remodelling.The presence of LV thrombus in 23% of the participants, which is a contraindication to the transendocardial procedure due to the risk of systemic arterial embolism and stroke, was another main reason for screen failure. This is consistent with expectations in large AMI34. The use of direct oral anticoagulants prior to transendocardial injection might overcome exclusions of these patients35.

[0251] Expansion of CD34+ cells in the EXCELLENT trial was comparable to that obtained from healthy subjects with the same manufacturing process (19±7.5-fold).24However, older age, co-morbidities and smoking status may negatively impact cell mobilization induced by G-CSF.36-39The mean number of injected cells was higher than the efficacy-associated threshold number reported in the PreSERVE-AMI study with intracoronary administrations of CD34+ cells to AMI patients.16Nevertheless, about 20% of participants randomised to the active group did not undergo transendocardial injections due to a low number of CD34+ cells at the beginning of the manufacturing process. In future trials, optimising the cell mobilization protocol in patients with expected low cell mobilization may need specific measures such as the use of drugs like plerixafor, known to mobilize CD34+ cells in various settings such as autologous stem cell transplantation.3839

[0252] Only one patient (3%) of the active group was hospitalized for heart failure during the 6-month follow up period, while during the same period five events were reported in three patients (19%) in the SoC group. Overall, the number of events and hospitalizations for heart failure was relatively low, a reflection of the effectiveness of contemporary guideline-directed medical therapy in patients with AMI. However, a significant proportion of patients do appear to require additional therapy to counteract adverse LV remodelling.

[0253] The trial was not powered to secondary outcome but positive congruent signals were observed on several individual surrogate markers40^6. The improvement in LVESVi observed at 3 months in both groups (-9% and -6% in the active and SoC groups respectively) seems to persist only in the active group over 6 months suggesting a sustained reverse remodelling. The increase in LVEDVi observed in the SoC group at 6 months (+9%) is consistent with the adverse remodelling previously highlighted in patients diagnosed with MVO.15The viability improvement has been observed in the areas with transmural scar or MVO. A paracrine stimulation of angiogenesis is considered to be a primary mechanism by which stem cells may attenuate and even reverse post-Ml remodeling4748. This effect could be all the more beneficial that the baseline perfusion isdramatically impaired by MVO. While the change in NT-ProBNP concentrations was exploratory and only available for two thirds of the trial population, the drop observed within the first months in the active group with a higher magnitude than in the SoC group is also encouraging.

[0254] We also acknowledge the potential independent effect of G-CSF administration in the active group which may have contributed to some of our findings. However, the largest available trial recently published was inconclusive and did not support the efficacy of the mobilization alone49.

[0255] In conclusion, we have shown that transendocardial injections of expanded autologous CD34+ cells in patients with recent large myocardial infarction was feasible. Some early signals of potential efficacy in LV remodelling, viability and NT-proBNP concentrations were observed and promising for long term benefits in subjects with severe post Ml and persistent high risk of developing heart failure.

[0256] Example 4: Phase 3 Clinical Study Protocol Synopsis

[0257] A Prospective Randomized, Concurrent-Controlled, Open-Label, Multicentre Phase 3 Study to Evaluate the Safety and Effectiveness of ProtheraCytes® Cardiac Therapy versus Standard of Care in Patients with Acute Myocardial Infarction

[0258] The finished product, ProtheraCytes®, is a fresh preparation for cell therapy that has the potential for regeneration of various damaged tissues, including cardiac tissue, following coronary artery occlusion.

[0259] The randomized Phase 1-2b EXCELLENT study (EUDRACT 2014-001476-63) has investigated the regeneration of damaged heart tissue after severe AMI to prevent the development of severe Chronic Heart Failure (CHF) in a population of 50 patients in France and in the UK. The study was completed in 2024. Patients received either the investigational product in addition to standard of care (SoC) or SoC alone. 34 subjects received ProtheraCytes®, and 16 subjects were enrolled in the SoC group.

[0260] ProtheraCytes®’s regenerative effect was observed as assessed by the change from baseline of two endpoints, i.e. the N-terminal pro-brain natriuretic peptide type B (NT- proBNP) dosage at Month 3 and the left ventricular end systolic volume index (LVESVi) as assessed by cMR at Month 6. Both endpoints are known to be associated with clinical outcomes such as “major adverse cardiovascular events” (MACE), comprising all death and heart failure-related (HF) hospitalization.Interestingly, ProtheraCytes® properties seem to have also a positive regenerative effect on infarcted zone with microvascular obstruction (MVO) as assessed by cMR and clinical outcome. Microvascular obstruction (MVO) is present in a subpopulation of patients with acute myocardial infarction. MVO usually arises following reperfusion therapy in patients after a prolonged period of severe myocardial ischemia. It is most commonly, but not exclusively, seen in patients with a delayed presentation to reperfusion treatment.

[0261] These patients are high-risk patients, known to have a higher risk of developing chronic heart failure.

[0262] MVO is characterized by damage and dysfunction of the myocardial microvasculature with a noreflow phenomenon within the infarct zone. While MVO may be demonstrated through several different imaging modalities, cardiac Magnetic Resonance (cMR) enables accurate identification of MVO. It also permits assessment of infarct extent and overall left ventricular function during the same imaging examination.

[0263] In the EXCELLENT study, viability data of the myocardium in the infarct zone measured by cMR suggest that ProtheraCytes® decrease the size of the infarct zone in the MVO areas.

[0264] The present clinical study will investigate the clinical benefit of ProtheraCytes® regenerative therapy via an adaptative design with an interim analysis to reevaluate the sample size if needed. The clinical benefit of ProtheraCytes® is assessed by generalized pairwise comparisons of five prioritized outcomes (in this order:)

[0265] (1) death from any cause within 24 months from randomization

[0266] (2) cardiovascular death within 24 months from randomization

[0267] (3) heart transplant or LVAD need within 24 months from randomization

[0268] (4) number of heart failure (HF) hospitalizations (HFH) or HFH equivalents (urgent HF visits) within 24 months from randomization

[0269] (5) % NT-pro-BNP change from baseline to 3 months, with a threshold of clinical relevance of 25%

[0270] (6) % change in Left Ventricle End Systolic Volume index (LVESVi) from baseline to 6 months, with a threshold of clinical relevance of 10%.This method of analysis will be employed both for an interim analysis that will take place when 80% of the patients are accrued, and for the final analysis that will take place 2 years after the last patient is accrued.

[0271] ProtheraCytes® are injected transendocardially via a catheter within 2- 3 months post-acute myocardial infarction (AMI). ProtheraCytes® administration consists of multiple injections in the infarcted area of the left ventricular wall. The procedure is minimally invasive.

[0272] The ProtheraCytes® optimal biological dose (OBD) was assessed considering tolerability and efficacy signals in the EXCELLENT study. The OBD is defined as the lowest well tolerated dose that provides the highest clinical benefit and that induces the desired regenerative effect in the infarcted zone.

[0273] ProtheraCytes® is a cell therapy to be administered ahead of severe Chronic Heart Failure (CHF) onset. CHF may lead to Cardiac Transplantation in high-risk patients, should a compatible graft be available in a timely manner. Only a small minority of patients have access to cardiac transplantation which is a costly procedure associated with a risk of graft rejection.

[0274] Hence, there is a high medical need for alternative therapy. CHF in high-risk patients is associated with high mortality, multiple complications, poor Quality of Life (QoL), and high health economic burden related to multiple hospitalizations.

[0275] In this proposed Phase 3 study, QoL is assessed via the 36-item Short-Form Health Survey (SF-36). The SF36 has good internal coherence and takes only a short time to answer the questions (approx. 15 min). In addition, 6-minute walk tests (6MWT) will be performed. The 6MWT (submaximal exercise test) may provide reliable information about the patient’s daily activity. Reduced functional ability and exercise tolerance in patients with HF are associated with poor quality of life and a poor prognosis. The 6MWT is a widely available and well-tolerated test for the assessment of the functional capacity of patients with HF. In the present study, health economic burden related to multiple hospitalization, cost of drug treatment related to heart condition and heart transplantation is assessed in selected clinical sites.

[0276] ProtheraCytes® are expected to demonstrate a safe profile and to address a strong unmet need related to AMI complications and CHF-related mortality in high-risk patients.

[0277] Objectives, Endpoints, and Estimands:Primary Efficacy: To assess ProtheraCytes® ’s effect on five prioritized outcomes of major clinical interest

[0278] Endpoints:

[0279] • death from any cause within 24 months from randomization

[0280] • cardiovascular death within 24 months from randomization

[0281] • heart transplantation or LVAD need within 24 months from randomization

[0282] • number of HF hospitalizations or HFH equivalents (urgent HF visits) within 24 months from randomization

[0283] • % NT-pro-BNP change from baseline to 3 months, with a threshold of clinical relevance of 25%

[0284] • % change in Left Ventricle End Systolic Volume index (LVESVi) from baseline to 6 months, with a threshold of clinical relevance of 10%.

[0285] Alpha-protected Secondary Efficacy: To assess ProtheraCytes® ’s effect on overall survival (OS) at Month 24

[0286] Endpoint: Overall Survival at Month 24

[0287] Other Secondary Efficacy:

[0288] To assess ProtheraCytes® ’s effect on recurrent myocardial infarction (Ml) Endpoint: Occurrence of recurrent myocardial infarction (Ml)

[0289] To assess ProtheraCytes® ’s effect on stroke

[0290] Endpoint: Occurrence of stroke

[0291] To assess ProtheraCytes® ’s effect on arrhythmias

[0292] Endpoint: Occurrence of arrhythmias

[0293] To assess ProtheraCytes® ’s effect on MACE incidence until Month 24.

[0294] Endpoint: Cumulative incidence of cardiac event related hospitalizations until Month 24 Overall Design:

[0295] This study is a prospective, multicentre, randomized, open label, concurrent-controlled, parallel group trial with a 24-month follow-up phase to be conducted in approximately 325 subjects with acute myocardial infarction.

[0296] Subjects will be stratified by age and by the presence of LV thrombus randomly assigned using an Interactive Voice / Web Response System (IxRS) to either ProtheraCytes® combined withStandard of Care or Standard of Care only (1:1 ratio). Mobilization is performed in ProtheraCytes® arm only. Mobilization regimen supports blood collection as source material for autologous regenerative cell therapy and is a safe and well-established procedure. The mobilization regimen consists of one daily subcutaneous injection of G-CSF (Lenograstim or filgrastim) and plerixafor. Lenograstim or filgrastim is administered at a dose of approximately 10 pg / kg per day for 5 days and plerixafor on the morning of Day 5, depending on the age of the subject. On day 5 of treatment with G-CSF, the patient will have 220 ml of whole blood harvest in the afternoon (+ / - 10 ml). After randomization all subjects start Standard of Care treatment for approximately 24 months, unless safety concerns requiring treatment interruption or discontinuation according to investigator’s judgment, or if predefined stopping criteria are met.

[0297] G-CSF daily injections may be fractionated to improve mobilization efficacy. The 1st, 4th and 5th G-CSF injections must be performed at the hospital by a qualified nurse. The 1st injection will assess the potential for allergic reaction. The 4th and 5th injections will assess leucocytosis. Regarding the 2nd and 3rd injections, at the discretion of the investigator and according to the patient’s health status, the patient could be discharged with the requisite number of G-CSF batches allowance for administration at home by a qualified nurse according to the schedule. These conditions are set to manage the appropriate leucocytosis ahead of 1st, 4th and 5th G-CSF administration. If White Blood Cell count at the 4th or 5th day exceeds 70 x 109cell / L, the corresponding G-CSF administration must be suspended.

[0298] ProtheraCytes® will be administered via trans-endocardial injection to subjects in the ProtheraCytes® therapy arm within 3 months following the day of the AMI onset.

[0299] Prior to intraventricular injection of the ProtheraCytes®, subjects will undergo a pre-injection control visit including cMR for measuring scar thickness, border zone and to confirm the absence of left ventricular thrombus.

[0300] The control patients will have the same visit schedule and follow-up as in the ProtheraCytes® arm. cMR is scheduled according to simulation at enrolment in the control arm.

[0301] Echocardiography and NT-proBNP testing will be performed at baseline, Months 1, 3, 6, 12, 18 and 24 visits.cMR will be performed at baseline, pre-injection, 6 months, and 24 months visits. Echocardiography and cMR will be conducted via centralized blinded and random reading.

[0302] In both randomized arms, through the end of the study, the patient may receive adapted oral and / or parenteral cardiac medications (not restricted to unfractionated Heparin or Low Molecular Weight Heparin) according to the patient’s status. However continuous use of i.v. catecholamines, mechanical hemodynamic support (aortic balloon pump) is not allowed if initiated 24 hours before screening cMR.

[0303] Other medications may be prescribed if patients experience non-severe ventricular arrhythmia or need anti-coagulants.

[0304] The interim analysis will occur when 80% of the patients are accrued. The estimate of 6 months follow-up at the time of interim analysis is approximately 50% of the final population of subjects in the study.

[0305] The interim analysis may enable the study to adapt the sample size to increase the recruitment by an extra 100 subjects if needed before the end of the accrual.

[0306] The final analysis will occur when all subjects have completed the Month 24 visit (EOS).

[0307] Brief Summary:

[0308] This study will evaluate the efficacy and safety of ProtheraCytes® combined with SoC compared with SoC alone as cardiac regenerative treatment in patients with AMI.

[0309] SoC may include non-investigational medicinal products in the following therapeutic classes:

[0310] • Mineralocorticoid Receptor Antagonists (MRA)

[0311] • Diuretics

[0312] • Angiotensin Conversion Enzyme inhibitors (ACEi)

[0313] • Angiotensin receptor blockers (ARB)

[0314] • Angiotensin receptor neprilysin inhibitor (ARNI) such as Sacubitril combined with ARB (valsartan)

[0315] • Sodium-Glucose Transport Protein 2 (SGLT2) Inhibitors,

[0316] • Statin,

[0317] • Platelet aggregation• Anticoagulants.

[0318] Cardiac regenerative treatment is a pre-emptive therapy to be administered to patients with MVO before onset of severe CHF. CHF may lead to serious complications, poor Quality of Life and short survival, or Cardiac Transplantation for a limited number of patients for whom a compatible graft is available in a timely manner.

[0319] Study Details include:

[0320] • The study duration will be approximately 48 months

[0321] • The duration of observation for each subject is approximately 25 months with 4 phases:

[0322] • Pre-Screening Day-40 or earlier to Day-30 (Typically Day-40 is Day+2±2 after last percutaneous coronary intervention or hospital presentation when no stent is implanted) • Screening from Day -30 to Day -20 and enrolment phase (randomization on Days -18 to- 16)

[0323] • Treatment phase (Day -15 to Day+2)

[0324] • Follow-up phase (Day+3 to Month +24)

[0325] • In the ProtheraCytes® arm, the study treatment will include trans-endocardial injection on Day 0 in addition to Standard of Care.

[0326] • IMP is ProtheraCytes®. Non-investigational medicinal products (NIMPs) include products used for mobilization and for Standard of Care of cardiovascular condition. Study treatment refers to both IMP and NIMP in this study.

[0327] • Subjects will be hospitalized for the administration of ProtheraCytes® (Day 0). The length of hospitalization will depend on the subject’s condition, investigator’s judgment, and local practices. The expected duration of hospitalization is 24 hours for monitoring of potential adverse effect of injection procedure.

[0328] • The study consists of the following visits:

[0329] • Pre-screening on Day-40 to Day-30

[0330] • Screening and randomization: Day -30 to Day -16 (randomization on Days -18 to Day-16) • Treatment

[0331] • Day -15 ± 2 (SoC safety and efficacy assessment in IMP arm)

[0332] • Day -11 ± 2 (blood collection for ProtheraCytes® manufacturing in IMP arm, SoC safety and efficacy assessment in IMP arm, schedule of event simulation in the SoC arm)• Day -4± 2 (Pre-injection visit in both arms, actual or according to event simulation, shipping of ProtheraCytes® and injection kit to the clinical site in IMP arm, safety and efficacy assessment in both arms)

[0333] • Day 0 (ProtheraCytes® trans-endocardial injection, safety and efficacy assessment in IMP arm)

[0334] • Day +1 safety and efficacy assessment in IMP arm (End of Treatment [EOT])

[0335] • Follow-up:

[0336] • Day 14 ± 2 days

[0337] • Day 30 ± 2 days

[0338] • Day 60 ± 3 days

[0339] • Month 3 ± 1 week

[0340] • Month 6 ± 1 week

[0341] • Month 12 ± 2 week

[0342] • Month 18 ± 2 week

[0343] • Month 24 ± 2 week (End of Study [EOS])

[0344] • Unscheduled visits may be arranged when deemed necessary by the

[0345] investigator or study medical monitor.

[0346] The duration of the study is roughly 48 months with an accrual period of 24 months, and an additional period of follow-up of 24 months.

[0347] Study Population:

[0348] Inclusion Criteria:

[0349] Type of Subject and Disease Characteristics

[0350] • Subjects with a diagnosis of AMI according to the AHA classification of AMI

[0351] with infarct on Left Ventricular sections

[0352] • Subjects with rise of troponin with at least one value 70 times above the upper reference limit.

[0353] • Subjects with a diagnosis of AMI within 1 week (7 days) after first symptoms, Day 0 being the day of last percutaneous coronary intervention or; Day 0

[0354] being the day of hospital presentation if no stent has been implanted.

[0355] • Combination of LVEF < 45% and LV akinetic or dyskinetic segment(s) - by Echography at D2 (+ / -2) as per local practice• Subjects with LVEF remaining < 45% as assessed by cMR at Day+8 (± 3) • Subjects with MVO as assessed by cMR at D8 (± 3) and >50% transmural extend of the infarct.

[0356] • Life expectancy > 6 months at screening.

[0357] • OMS Performance Status < 2.

[0358] References

[0359] 1. Harrington J, Jones WS, Udell JA, Hannan K, Bhatt DL, Anker SD, Petrie MC, Vedin O, Butler J, Hernandez AF. Acute Decompensated Heart Failure in the Setting of Acute Coronary Syndrome. JACC Heart Fail 2022;10:404-414.

[0360] 2. Salari N, Morddarvanjoghi F, Abdolmaleki A, Rasoulpoor S, Khaleghi AA, Hezarkhani LA, Shohaimi S, Mohammadi M. The global prevalence of myocardial infarction: a systematic review and meta-analysis. BMC Cardiovasc Disord 2023,2320Q.

[0361] 3. Mehran R, Steg PG, Pfeffer MA, Jering K, Claggett B, Lewis EF, Granger C, Kober L, Maggioni A, Mann DL, McMurray JJV, Rouleau J-L, Solomon SD, Ducrocq G, Berwanger O, De Pasquale CG, Landmesser U, Petrie M, Leng DSK, Van Der Meer P, Lefkowitz M, Zhou Y, Braunwald E. The Effects of Angiotensin Receptor-Neprilysin Inhibition on Major Coronary Events in Patients With Acute Myocardial Infarction: Insights From the PARADISE-MI Trial. Circulation 2022;146:1749-1757.

[0362] 4. Butler J, Jones WS, Udell JA, Anker SD, Petrie MC, Harrington J, Mattheus M, Zwiener I, Amir O, Bahit MC, Bauersachs J, Bayes-Genis A, Chen Y, Chopra VK, Figtree G, Ge J, Goodman SG, Gotcheva N, Goto S, GasiorT, Jamal W, Januzzi JL, Jeong MH, Lopatin Y, Lopes RD, Merkely B, Parikh PB, Parkhomenko A, Ponikowski P, Rossello X, Schou M, Simic D, Steg PG, Szachniewicz J, Meer P van der, Vinereanu D, Zieroth S, Brueckmann M, Sumin M, Bhatt DL, Hernandez AF. Empagliflozin after Acute Myocardial Infarction. N Engl J Med 2024;390:1455-1466.

[0363] 5. James S, Erlinge D, Storey RF, McGuire DK, Beider M de, Eriksson N, Andersen K, Austin D, Arefalk G, Carrick D, Hofmann R, Hoole SP, Jones DA, Lee K, Tygesen H, Johansson PA, Langkilde AM, Ridderstrale W, Parvaresh Rizi E, Deanfield J, Oldgren J. Dapagliflozin in Myocardial Infarction without Diabetes or Heart Failure. NEJM Evid 2024;3:EVIDoa2300286.

[0364] 6. Symons R, Pontone G, Schwitter J, Francone M, Iglesias JF, Barison A, Zalewski J, De Luca L, Degrauwe S, Claus P, Guglielmo M, Nessler J, Carbone I, Ferro G, Durak M, Magistrelli P, Lo Presti A, Aquaro GD, Eeckhout E, Roguelov C, Andreini D, Vogt P, Guaricci Al, Mushtaq S, Lorenzoni V, Muller O, Desmet W, Agati L, Janssens S, Bogaert J, Masci PG. Long-Term Incremental Prognostic Value of Cardiovascular Magnetic Resonance After ST-Segment Elevation Myocardial Infarction. JACC: Cardiovascular Imaging 2018;11:813-825.7. Pfeffer MA, Claggett B, Lewis EF, Granger CB, Kober L, Maggioni AP, Mann DL, McMurray JJV, Rouleau J-L, Solomon SD, Steg PG, Berwanger O, Cikes M, De Pasquale CG, East C, Fernandez A, Jering K, Landmesser U, Mehran R, Merkely B, Vaghaiwalla Mody F, Petrie MC, Petrov I, Schou M, Senni M, Sim D, Van Der Meer P, Lefkowitz M, Zhou Y, Gong J, Braunwald E. Angiotensin Receptor-Neprilysin Inhibition in Acute Myocardial Infarction. N Engl J Med 2021;385:1845-1855.

[0365] 8. Delewi R, Hirsch A, Tijssen JG, Schachinger V, Wojakowski W, Roncalli J, Aakhus S, Erbs S, Assmus B, Tendera M, Goekmen Turan R, Corti R, Henry T, Lemarchand P, Lunde K, Cao F, Huikuri HV, Surder D, Simari RD, Janssens S, Wollert KC, Plewka M, Grajek S, Traverse JH, Zijlstra F, Piek JJ. Impact of intracoronary bone marrow cell therapy on left ventricular function in the setting of ST-segment elevation myocardial infarction: a collaborative meta-analysis. European Heart Journal 2014;35:989-998.

[0366] 9. Parizadeh SM, Jafarzadeh-Esfehani R, Ghandehari M, Parizadeh MR, Ferns GA, Avan A, Hassanian SM. Stem cell therapy: A novel approach for myocardial infarction. Journal Cellular Physiology 2019;234: 16904-16912.

[0367] 10. Roncalli JG, Tongers J, Renault M-A, Losordo DW. Endothelial progenitor cells in regenerative medicine and cancer: a decade of research. Trends in Biotechnology 2008;26:276-283.

[0368] 11. Tongers J, Roncalli JG, Losordo DW. Role of endothelial progenitor cells during ischemia- induced vasculogenesis and collateral formation. Microvascular Research 2010;79:200- 206.

[0369] 12. Lamirault G, Susen S, Forest V, Hemont C, Parini A, Corvoisier PL, Piot C, Richard M-J, Delasalle B, Rouard H, Sportouch C, Persoons V, Van Belle E, Roncalli J, Lemarchand P. Difference in mobilization of progenitor cells after myocardial infarction in smoking versus non-smoking patients: insights from the BONAMI trial. Stem Cell Res Ther 2013;4: 152. 13. Tendera M, Wojakowski W, Ruzyllo W, Chojnowska L, K^pka C, Tracz W, Musialek P, Piwowarska W, Nessler J, Buszman P, Grajek S, Br^borowicz P, Majka M, Ratajczak MZ. Intracoronary infusion of bone marrow-derived selected CD34+CXCR4+ cells and nonselected mononuclear cells in patients with acute STEMI and reduced left ventricular ejection fraction: results of randomized, multicentre Myocardial Regeneration by Intracoronary Infusion of Selected Population of Stem Cells in Acute Myocardial Infarction (REGENT) Trial. European Heart Journal 2009;30:1313-1321.

[0370] 14. Martin-Rendon E, Brunskill SJ, Hyde CJ, Stanworth SJ, Mathur A, Watt SM. Autologous bone marrow stem cells to treat acute myocardial infarction: a systematic review. European Heart Journal 2008;29: 1807-1818.

[0371] 15. Davidson SJ, Roncalli J, Surder D, Corti R, Chugh AR, Yang PC, Henry TD, Stanberry L, Lemarchand P, Beregi J-P, Traverse JH. Microvascular obstruction identifies a subgroup of patients who benefit from stem cell therapy following ST-elevation myocardial infarction. American Heart Journal 2023;259:79-86.

[0372] 16. Quyyumi AA, Vasquez A, Kereiakes DJ, Klapholz M, Schaer GL, Abdel-Latif A, Frohwein S, Henry TD, Schatz RA, Dib N, Toma C, Davidson CJ, Barsness GW, Shavelle DM,Cohen M, Poole J, Moss T, Hyde P, Kanakaraj AM, Druker V, Chung A, Junge C, Preti RA, Smith RL, Mazzo DJ, Pecora A, Losordo DW. PreSERVE-AMI: A Randomized, DoubleBlind, Placebo-Controlled Clinical Trial of Intracoronary Administration of Autologous CD34+ Cells in Patients With Left Ventricular Dysfunction Post STEMI. Circ Res 2017;120:324-331.

[0373] 17. Quyyumi AA, Waller EK, Murrow J, Esteves F, Galt J, Oshinski J, Lerakis S, Sher S, Vaughan D, Perin E, Willerson J, Kereiakes D, Gersh BJ, Gregory D, Werner A, Moss T, Chan WS, Preti R, Pecora AL. CD34+ cell infusion after ST elevation myocardial infarction is associated with improved perfusion and is dose dependent. American Heart Journal 2011;161:98-105.

[0374] 18. Vrtovec B, Poglajen G, Sever M, Zemljic G, Frljak S, Cerar A, Cukjati M, Jaklic M, Cernelc P, Haddad F, Wu JC. Effects of Repetitive Transendocardial CD34+Cell Transplantation in Patients With Nonischemic Dilated Cardiomyopathy. Circ Res 2018;123:389-396.

[0375] 19. Hunt SA. ACC / AHA 2005 Guideline Update for the Diagnosis and Management of Chronic Heart Failure in the Adult. Journal of the American College of Cardiology 2005 ;46:e1-e82.

[0376] 20. Duong HK, Savani BN, Copelan E, Devine S, Costa LJ, Wingard JR, Shaughnessy P, Majhail N, Perales M-A, Cutler CS, Bensinger W, Litzow MR, Mohty M, Champlin RE, Leather H, Giralt S, Carpenter PA. Peripheral Blood Progenitor Cell Mobilization for Autologous and Allogeneic Hematopoietic Cell Transplantation: Guidelines from the American Society for Blood and Marrow Transplantation. Biology of Blood and Marrow Transplantation 2014;20: 1262-1273.

[0377] 21. Bastani S, Staal FJT, Cante-Barrett K. The quest for the holy grail: overcoming challenges in expanding human hematopoietic stem cells for clinical use. Stem Cell Investig 2023;10:15.

[0378] 22. Panch SR, Szymanski J, Savani BN, Stroncek DF. Sources of Hematopoietic Stem and Progenitor Cells and Methods to Optimize Yields for Clinical Cell Therapy. Biol Blood Marrow Transplant 2017;23: 1241 -1249.

[0379] 23. Sakurai M, Ishitsuka K, Becker HJ, Yamazaki S. Ex vivo expansion of human hematopoietic stem cells and clinical applications. Cancer Sci 2024;115:698-705.

[0380] 24. Saucourt C, Vogt S, Merlin A, Valat C, Criquet A, Harmand L, Birebent B, Rouard H, Himmelspach C, Jeandidier E, Chartois-Leaute A-G, Derenne S, Koehl L, Salem J-E, Hulot J-S, Tancredi C, Aries A, Jude S, Martel E, Richard S, Douay L, Henon P. Design and Validation of an Automated Process for the Expansion of Peripheral Blood-Derived CD34+ Cells for Clinical Use After Myocardial Infarction. Stem Cells Translational Medicine 2019;8:822-832.

[0381] 25. Byrne RA, Rossello X, Goughian J J, Barbate E, Berry C, Chieffo A, Claeys MJ, Dan G-A, Dweck MR, Galbraith M, Gilard M, Hinterbuchner L, Jankowska EA, Juni P, Kimura T, Kunadian V, Leosdottir M, Lorusso R, Pedretti RFE, Rigopoulos AG, Rubini Gimenez M, Thiele H, Vranckx P, Wassmann S, Wenger NK, Ibanez B, ESC Scientific Document Group, Halvorsen S, James S, Abdelhamid M, Aboyans V, Marsan NA, Antoniou S, Asteggiano R, Back M, Capodanno D, Casado-Arroyo R, Cassese S, Celutkiene J, CikesM, Collet J-P, Ducrocq G, FalkV, Fauchier L, Geisler T, Gorog DA, Holmvang L, Jaarsma T, Jones HW, Kober L, Koskinas KC, Kotecha D, Krychtiuk KA, Landmesser U, Lazaros G, Lewis BS, Lindahl B, Linhart A, Lochen M-L, Mamas MA, McEvoy JW, Mihaylova B, Mindham R, Mueller C, Neubeck L, Niebauer J, Nielsen JC, NiessnerA, Paradies V, Pasquet AA, Petersen SE, Prescott E, Rakisheva A, Rocca B, Rosano GMC, Sade LE, Schiele F, Siller-Matula JM, Sticherling C, Storey RF, Thielmann M, Vrints C, Windecker S, Wiseth R, Witkowski A, El Amine Bouzid M, Hayrapetyan H, Metzler B, Lancellotti P, Bajric M, Karamfiloff K, Mitsis A, Ostadal P, Sorensen R, Elwasify T, Marandi T, Rybdi E, Collet J-P, Chukhrukidze A, Mehilli J, Davlouros P, Becker D, Gudmundsdottir IJ, Crowley J, Abramowitz Y, Indolfi C, Sakhov O, Elezi S, Beishenkulov M, Erglis A, Moussallem N, Beniamin H, Dobiliene O, Degrell P, Balbi MM, Grosu A, Lakhal Z, Ten Berg J, Pejkov H, Angel K, Witkowski A, De Sousa Almeida M, Chioncel O, Bertelli L, Stojkovic S, Studencan M, Radsel P, Ferreiro JL, Ravn-Fischer A, Raber L, Marjeh MYB, Hassine M, Yildirir A, Parkhomenko A, Banning AP, Prescott E, James S, Arbelo E, Baigent C, Borger MA, Buccheri S, Ibanez B, Kober L, Koskinas KC, McEvoy JW, Mihaylova B, Mindham R, Neubeck L, Nielsen JC, Pasquet AA, Rakisheva A, Rocca B, Rossello X, Vaartjes I, Vrints C, Witkowski A, Zeppenfeld K. 2023 ESC Guidelines for the management of acute coronary syndromes. European Heart Journal 2023;44:3720-3826.

[0382] 26. Hamirani YS, Wong A, Kramer CM, Salerno M. Effect of Microvascular Obstruction and Intramyocardial Hemorrhage by CMR on LV Remodeling and Outcomes After Myocardial Infarction. JACC: Cardiovascular Imaging 2014;7:940-952.

[0383] 27. Wu E, Judd RM, Vargas JD, Klocke FJ, Bonow RO, Kim RJ. Visualisation of presence, location, and transmural extent of healed Q-wave and non-Q-wave myocardial infarction. The Lancet 2001 ; 357:21-28.

[0384] 28. Roncalli J, Roubille F, Meyer N, Pompilio G, Leroux L, Henon P, Trebuchet G, Criquet A, Kalbermatten M de, Saloux E, Manrique A, Marie P-Y, Bhatt DL, Solomon SD, Montalescot G, Newby DE, Zannad F, EXCELLENT Trial Investigators. Transendocardial injection of expanded autologous CD34+ cells after myocardial infarction: Design of the EXCELLENT trial. ESC Heart Fail 2024.

[0385] 29. Anderson HV (“Skip”), Masri SC, Abdallah MS, Chang AM, Cohen MG, Elgendy IY, Gulati M, LaPoint K, Madan N, Moussa ID, Ramirez J, Simon AW, Singh V, Waldo SW, Williams MS. 2022 ACC / AHA Key Data Elements and Definitions for Chest Pain and Acute Myocardial Infarction. Journal of the American College of Cardiology 2022;80:1660-1700.

[0386] 30. Dib N, Menasche P, Bartunek J J , Zeiher AM, Terzic A, Chronos NA, Henry TD, Peters NS, Fernandez-Aviles F, Yacoub M, Sanborn TA, DeMaria A, Schatz RA, Taylor DA, Fuchs S, Itescu S, Miller LW, Dinsmore JH, Dangas GD, Popma J J, Hall JL, Holmes DR.

[0387] Recommendations for Successful Training on Methods of Delivery of Biologies for Cardiac Regeneration. JACC: Cardiovascular Interventions 2010;3:265-275.

[0388] 31. Huber A, Oldridge N, Hofer S. International SF-36 reference values in patients with ischemic heart disease. Qua! Life Res 2016;25:2787-2798.

[0389] 32. Hesse B, Tagil K, Cuocolo A, Anagnostopoulos C, Bardies M, Bax J, Bengel F, Busemann Sokole E, Davies G, Dondi M, Edenbrandt L, Franken P, Kjaer A, Knuuti J, Lassmann M, Ljungberg M, Marcassa C, Marie PY, McKiddie F, O’Connor M, Prvulovich E, UnderwoodR, Van Eck-Smit B. EANM / ESC procedural guidelines for myocardial perfusion imaging in nuclear cardiology. EurJ Nucl Med Mol Imaging 2005;32:855-897.

[0390] 33. Heusch G. Myocardial stunning and hibernation revisited. Nat Rev Cardiol 2021; 18:522- 536.

[0391] 34. Delewi R, Nijveldt R, Hirsch A, Marcu CB, Robbers L, Hassell MECJ, Bruin RHA de, Vleugels J, Laan AM van der, Bouma BJ, Tio RA, Tijssen JGP, Rossum AC van, Zijlstra F, Piek JJ. Left ventricular thrombus formation after acute myocardial infarction as assessed by cardiovascular magnetic resonance imaging. EurJ Radiol 2012;81:3900-3904.

[0392] 35. Levine GN, McEvoy JW, Fang JC, Ibeh C, McCarthy CP, Misra A, Shah Zl, Shenoy C, Spinier SA, Vallurupalli S, Lip GYH, on behalf of the American Heart Association Council on Clinical Cardiology; Council on Cardiovascular and Stroke Nursing; and Stroke Council. Management of Patients at Risk for and With Left Ventricular Thrombus: A Scientific Statement From the American Heart Association. Circulation 2022;146.

[0393] 36. Wuchter P, Ran D, Bruckner T, Schmitt T, Witzens-Harig M, Neben K, Goldschmidt H, Ho AD. Poor Mobilization of Hematopoietic Stem Cells — Definitions, Incidence, Risk Factors, and Impact on Outcome of Autologous Transplantation. Biology of Blood and Marrow Transplantation 2010;16:490-499.

[0394] 37. Fadini GP, Albiero M, Vigili De Kreutzenberg S, Boscaro E, Cappellari R, Marescotti M, Poncina N, Agostini C, Avogaro A. Diabetes Impairs Stem Cell and Proangiogenic Cell Mobilization in Humans. Diabetes Care 2013;36:943-949.

[0395] 38. Bilgin YM, De Greef GE. Plerixafor for stem cell mobilization: the current status. Current Opinion in Hematology 2016; 23: 67 -71.

[0396] 39. Romon I, Castillo C, Cid J, Lozano M. Use of plerixafor to mobilize haematopoietic progenitor cells in healthy donors. Vox Sanguinis 2022;117:6-16.

[0397] 40. Kwon DH, Hachamovitch R, Popovic ZB, Starling RC, Desai MY, Flamm SD, Lytle BW, Marwick TH. Survival in Patients With Severe Ischemic Cardiomyopathy Undergoing Revascularization Versus Medical Therapy: Association With End-Systolic Volume and Viability. Circulation 2012;126.

[0398] 41. Funaro S, La Torre G, Madonna M, Galiuto L, Scara A, Labbadia A, Canali E, Mattatelli A, Fedele F, Alessandrini F, Crea F, Agati L, on behalf of AMICI Investigators. Incidence, determinants, and prognostic value of reverse left ventricular remodelling after primary percutaneous coronary intervention: results of the Acute Myocardial Infarction Contrast Imaging (AMICI) multicenter study. European Heart Journal 2009;30:566-575.

[0399] 42. Gold MR, Daubert C, Abraham WT, Ghio S, St. John Sutton M, Hudnall JH, Cerkvenik J, Linde C. The effect of reverse remodeling on long-term survival in mildly symptomatic patients with heart failure receiving cardiac resynchronization therapy: Results of the REVERSE study. Heart Rhythm 2015;12:524-530.

[0400] 43. Montalescot G, Pitt B, Lopez De Sa E, Hamm CW, Flather M, Verheugt F, Shi H, Turgonyi E, Orri M, Vincent J, Zannad F, for the REMINDER Investigators, Noll G, Weir R, O’Neill B,Bohm M, Hillis WS, Grieve A, Rouleau J-L, Gerasimos F, Fitchett D, Lepage S, Madan M, Sussex B, Tremblay G, Welsh R, Wong G, Hutyra M, Kettner J, Ostadal P, Spinar J, Vojacek J, Barboteu M, Collet J-P, Coste P, Cottin Y, Ducos D, Galinier M, Teiger E, Zemour G, Bauersachs J, Hambrecht R, Hauf G, Heuer H, Mudra H, Munzel T, Steiner S, Strasser R, Sydow K, Tschope C, Wachter R, Werner N, Alexopoulos D, Babalis D, Pyrgakis V, Dezsi C, Lupkovics G, Polgar P, Tomcsanyi J, Herrman J, Ten Berg JM, Gorny J, Kubica J, Lewczuk J, Zmuda W, Hranai M, Kovar F, Margoczy R, Micko K, Sumbal J, Genover XB, Ortiz AF, Sala MF, Garcia CG, Munoz CP, Rey Blas JR, Soriano FR, Adamson D, Alamgir F, Chauhan A, Lip G, Martin T, McCann G, Newby D, Smith D. Early eplerenone treatment in patients with acute ST-elevation myocardial infarction without heart failure: The Randomized Double-Blind Reminder Study. European Heart Journal 2014;35:2295-2302.

[0401] 44. De Waha S, Patel MR, Granger CB, Ohman EM, Maehara A, Eitel I, Ben-Yehuda O, Jenkins P, Thiele H, Stone GW. Relationship between microvascular obstruction and adverse events following primary percutaneous coronary intervention for ST-segment elevation myocardial infarction: an individual patient data pooled analysis from seven randomized trials. European Heart Journal 2017;38:3502-3510.

[0402] 45. Olivier A, Girerd N, Michel JB, Ketelslegers JM, Fay R, Vincent J, Bramlage P, Pitt B, Zannad F, Rossignol P, EPHESUS Investigators. Combined baseline and one-month changes in big endothelin-1 and brain natriuretic peptide plasma concentrations predict clinical outcomes in patients with left ventricular dysfunction after acute myocardial infarction: Insights from the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS) study. Int J Cardiol 2017;241:344-350.

[0403] 46. Stone GW, Selker HP, Thiele H, Patel MR, Udelson JE, Ohman EM, Maehara A, Eitel I, Granger CB, Jenkins PL, Nichols M, Ben-Yehuda O. Relationship Between Infarct Size and Outcomes Following Primary PCI: Patient-Level Analysis From 10 Randomized Trials. J Am Coll Cardiol 2016;67:1674-1683.

[0404] 47. Banerjee MN, Belli R, Hare JM. Clinical Studies of Cell Therapy in Cardiovascular Medicine: Recent Developments and Future Directions. Circ Res 2018;123:266-287.

[0405] 48. Aries A, Vignon C, Zanetti C, Goubaud A, Cormier A, Diederichs A, Lahlil R, Henon P, Garitaonandia I. Development of a potency assay for CD34+ cell-based therapy. Sci Rep 2023;13:19665.

[0406] 49. Achilli F, Maggiolini S, Madotto F, Bassetti B, Gentile F, Maggioni AP, Colombo Gl, Pompilio G, STEM-AMI OUTCOME Trial Investigators. Granulocyte colony-stimulating factor for stem cell mobilisation in acute myocardial infarction: a randomised controlled trial. Heart 2024; 110:1316-1326.

Claims

CLAIMS1. A population of CD34+ cells, for use in a cardiac regenerative therapy to reduce the development of chronic heart failure in severe post-acute myocardial infarction (AMI) subject, wherein said cardiac regenerative therapy comprises transendocardial injection of an efficient amount of said population of CD34+ cells in the left ventricular wall,wherein said subject is selected among the subjects with severe AMI characterized by (i) akinetic or dyskinetic left ventricular segments and Left Ventricular Ejection Fraction (LVEF) below 45% at day 2 (±2)(ii) Left Ventricular Ejection Fraction (LVEF) remaining below 45% at day +8 (±3), (iii) microvascular obstruction (MVO) at day +8 (±3),(iv) non-viable segment characterized by transmural extent of the infarct above 50%, at day +8 (±3), and / or,(v) a troponin peak with at least one value 70 times above the upper reference limit between day 0 and day 8,day 0 being the day of last percutaneous coronary intervention or day of hospitalization if no stent has been implanted.

2. The population of CD34+ cells, for use of Claim 1 ,wherein said subject is selected among the subjects with severe AMI characterized by (i) akinetic or dyskinetic left ventricular segments and Left Ventricular Ejection Fraction (LVEF) below 45% at day 2 (±2)(ii) Left Ventricular Ejection Fraction (LVEF) below 45% at day +8 (±3),(iii) microvascular obstruction (MVO) at day +8 (±3),(iv) above 50% transmural extent of the infarct, or akinetic or dyskinetic segments, at day +8 (±3), and,(v) a troponin peak with at least one value 70 times above the upper reference limit between day 0 and day 8.

3. The population of CD34+ cells for use according to Claim 1 or 2, wherein said population of CD34+ cells are administered within 3 months, for example within the second month, following the day of the AMI onset.

4. The population of CD34+ cells for use according to any one of Claims 1 - 3, wherein said CD34+ cells are expanded CD34+ cells, preferably autologous expanded CD34+ cells.

5. The population of CD34+ cells for use according to any one of Claims 1 - 4, wherein said population of CD34+ cells is obtained from peripheral blood mononuclear cells of said subject, after mobilization.

6. The population of CD34+ cells for use according to Claim 5, wherein mobilization comprises daily administration of G-CSF for 5 days with a dose of 10pg / kg / day before cell harvest, and, optionally, in combination with plerixafor on day 5 of administration of G-CSF.

7. The population of CD34+ cells for use according to any of Claims 1 - 6, wherein said population of CD34+ cells comprises at least 76% of viable CD34+ cells.

8. The population of CD34+ cells for use according to any one of Claims 1 - 7, wherein said CD34+ cells are obtained by(i) selecting CD34+ cells by immunoselection from a peripheral blood cell sample of said subject,(ii) incubating said population of selected CD34+ cells and culturing in a culture medium for ex vivo expansion, and(iii) selecting CD34+ cells from the ex vivo expanded cells.

9. The population of CD34+ cells for use according to Claim 8, wherein the step of ex vivo expansion comprises culturing an initial amount of less than 13 x 106viable CD34+ cells for 5 to 15 days, for example about 9 days, in a CD34+ stem cell culture medium.

10. The population of CD34+ cells for use according to Claim 8 or 9, wherein the quantity of VEGF in the cell culture supernatant at the end of the ex vivo expansion phase ranges from 100 pg / mL to 2800 pg / mL.

11. The population of CD34+ cells, for use according to any one of Claims 7 - 10, wherein the expansion rate is comprised between 5 and 100, for example between 10 and 20.

12. The population of CD34+ cells, for use according to any one of Claims 1 - 11, wherein said population of CD34+ cells injected in said subject comprises at least 6 x 106CD34+ cells.

13. The population of CD34+ cells, for use according to any one of Claims 1 - 12, wherein said population of CD34+ cells comprises more than 76% CD34+ cells and more than 85% viable cells.

14. The population of CD34+ cells, for use according to any one of Claims 1 - 15, wherein said subject receives said cardiac regenerative therapy in addition to standard of care for chronic heart failure, wherein said standard of care includes administering one or more of the following medicinal products:Mineralocorticoid Receptor Antagonists,Diuretics,- Angiotensin Conversion Enzyme Inhibitors- Angiotensin receptor blockers,- Angiotensin receptor neprilysin inhibitor,- Sodium-Glucose Transport Protein 2 inhibitors,- Statins,Platelet aggregation and- anticoagulants.

15. The population of CD34+ cells, for use according to any one of Claims 1 - 16, wherein said cardiac regenerative therapy(i) promotes reverse remodelling of the left ventricle as assessed by a decrease in Left Ventricular Ejection Systolic Volume index (LVESVi) in the first 6 months posttreatment as compared to start of the treatment, for example a decrease of at least 10% at about 3 months to about 6 months as compared to start of treatment; (ii) induces a decrease in N-terminal pro-brain natriuretic peptide type B (NT-proBNP) levels in the first 6 months post-treatment as compared to start of the treatment, for example a decrease of at least 25% of NT-pro-BNP secretion at about 3 months as compared to start of treatment; and / or,(iii) reduces the infarcted zone as assessed by the number of transmural segments with late gadolinium enhancement >50%, or in particular >75%, in the first 6 months posttreatment.

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