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Activation of vasculature associated stem cells and muscle stem cells for the repair and maintenance of muscle tissue

Final Report Summary - ENDOSTEM (Activation of vasculature associated stem cells and muscle stem cells for the repair and maintenance of muscle tissue)

Executive Summary:
The overall objective of ENDOSTEM was to leverage European scientific excellence through a collaborative project in order to develop new strategies to activate and mobilize muscle tissue-associated endogenous stem cells as a tool for efficient tissue repair and as an alternative to stem cell transplantation. We have focused our research efforts on candidate compounds that target muscle and muscle vasculature progenitor cells as well as prevent a damage response. This effort has been complemented by bio-delivery strategies for effective targeting of muscle tissues. These molecules include nitric oxide associated with non¬ steroidal anti-¬inflammatory drugs, histone desacetylase inhibitor (Givinostat), Omigapil, HMGB1 and Cripto. Our project brought together leading investigators to examine how vascular and muscle progenitors participate in postnatal growth and muscle tissue repair. A key issue that this project addressed is how the tissue environment in which endogenous stem cells are activated influences the capacity of stem cells to regenerate the muscle. We have demonstrated that muscle degeneration and fibrosis provokes altered vascularization and immune responses, which affect negatively stem cell function. We have shown that molecules interacting with neighboring vascular, inflammatory and fibrotic cell types can be used to therapeutically target stem cells. This strategy lead to effective approaches to muscle regenerative medicine.
Most importantly, three clinical trials have been initiated during ENDOSTEM project and are still ongoing:
• Phase I Omigapil clinical trial is recruting patients for Congential Muscular Dystrophy
• Phase I/II Givinostat clinical trial is ongoing with Duchenne Muscluar Dystrophy patients
• Phase I/II NO-NSAID clinical trial is ongoing with Duchenne Muscluar Dystrophy patients

Some of these clinical trials have been performed in tandem with experimentation to dissect the underlying mechanisms of action. This approach allowed us to identify new and innovative targets for muscle regeneration therapies such as PW1 expressing interstitial cells or fibro-adipogenic progenitors cells.

Project Context and Objectives:
The overall objective of ENDOSTEM is to leverage European scientific excellence through a collaborative project in order to develop new strategies to activate and mobilize muscle tissue-associated endogenous stem cells as a tool for efficient tissue repair and as an alternative to stem cell transplantation. We have focused our research efforts on candidate compounds that target muscle and muscle vasculature progenitor cells as well as prevent a damage response. This effort has been complemented by bio-delivery strategies for effective targeting of muscle tissues. This synergistic approach ensured that ongoing and future clinical trials have a greater chance of success.
To this end, key actions have been identified:

1. Phase I and Phase IIa clinical trials for patients with muscular disorders.
2. Characterization of actual target cells in the muscle repair processes in order to design future therapeutic approaches, which can also be applied to other tissues.
3. Analysis of neo-vascularisation, a key success factor in all efficient tissue regeneration.
4. Immunological studies both in vitro and in vivo in order to determine (i) any adverse or beneficial effect and (ii) factors that can modulate the immune response.
5. Stem cells fate and nuclear reprogramming analysis using epigenetic modifiers.
6. Refinement of bio-delivery of selected compounds.
7. Characterisation of (i) regulatory, (ii) transcriptional and (iii) signalling pathways that control stem cell activity in large animal models

To achieve these stated goals, the programme of work focuses on six interrelated projects in which the partners bring in specific expertise and resources that are collectively leveraged.
The primary focus was on clinical trials (CT) with selected pharmaceutical agents. CT represents the core of the ENDOSTEM project: they had benefit from discoveries during preclinical trials in animal models and their results have lead to the identification of new therapeutic approaches.
Four clinical trials are initiated (i) Phase IIa Clinical trial using Nitric Oxide (NO) in combination with anti-inflammatory agents (ii) Phase Ib/IIa Clinical trial using an HDAC inhibitor (iiii) Phase I clinical trial using Omigapil, a small anti-apoptotic molecule. These agents serve as critical tools to dissect muscle and muscle vasculature stem cell biology and promise to generate therapeutic agents for chronic degenerative vascular and muscle diseases.

The second project focused on discovering and understanding new signalling pathways that control muscle stem cells fate and regeneration. As the muscle satellite cell is an obvious cellular target, one of the aims of our project was to understand how satellite cell activation is controled, how they enter into cell cycle quiescence (Go) and whether and how cell fate choices are made. Simply put, it was critical to pursue the fundamental characterization of the actual target cells in the muscle repair process in order to intelligently design future therapeutic approaches. In addition, other muscle resident cell populations such as the PICs (see below) or ‘circulating’ stem cells have been shown to participate in muscle regeneration under experimental conditions, during these five years, it was fundamental to demonstrate the involvement of these newly carachterized stem cells in muscle regenration. This part of the project relies on results produced in small animals and constitutes a key translational interface between stem cell biology and the implementation of clinical protocols.
The third focus was to understand the two ways in which endogenous vascular progenitors contribute to muscle regeneration. The first is by induction of new vessels and therefore by increasing tissue perfusion and oxygenation. The second is through differentiation in new muscle fibers. We have studied the best conditions to improve both processes aimed to bring new adjuvant therapies to muscle regeneration. In this objective defined the best strategies to increase the number of these cells in the regenerating muscle and to obtain optimal conditions for muscular recovery by focusing on (i) the improvement of circulating vascular associated progenitors homing in the damaged tissue, to both contribute to muscle regeneration and neo-angiogenesis (ii) the induction of vascular stability to optimize muscle perfusion. In this regard, strategies aimed to increase circulating vascular progenitors homing and mobilization can be increased both by chemokines and by increasing cell adhesion and mobility through the endothelial cell monolayer.
The fourth focus was on the immunological aspects that result from tissue damage and invasion of muscle by inflammatory cells. Tissue damage and cell necrosis initiate the rapid and sequential invasion of muscle by inflammatory cells, such as leukocytes and macrophages. Chemokines are secreted by damaged or responding cells that affect the tissue milieu and affect the repair process. This family of structurally related chemotactic cytokines directs the migration of leukocytes throughout the body as well as epithelial and endothelial cells. We have shown that stem cells are also affected in this process. Recruited leucocytes play crucial, although apparently contrasting, roles: they are required for activation and differentiation of precursor cells and for matrix assembly. At the same time they actively amplify the damage. Particularly, we have focused our attention on macrophages and aimed to understand how these cells are influencing the course of muscle injury and regeneration in mouse models as well as in human biopsies.
The fifth project focused on new pharmacological approaches and delivery mechanisms. Stem cell lineage commitment to specialized cell types is driven by progressive chromatin changes that restrict the accessibility to transcription machinery, resulting in the selective expression of discrete sub¬sets of genes. An understanding of the mechanisms underlying the global and focal changes in chromatin structure – the stem cell nuclear reprogramming is essential to devise pharmacological interventions that can manipulate expression of key genes to promote stem cell- based repair of injured or diseased organs and tissues. In this regard, we have focused our work on studying the relation between stem cell fate and nuclear reprogramming by analysis and intervention on the epigenetic state of some key genes that could promote stem cell based repair. In addition, strategies for efficient targeting of muscle tissue using biomaterials approaches are developed.

Finally, the sixth goal was the preclinical testing of therapies for skeletal muscles diseases in clinically relevant large animal models. It is a well known that therapy testing in mouse models of human diseases, although a necessary step before clinical testing, is not an accurate predictor of the human response. Sometimes the different responses are due to unknown or poorly understood biological differences between the two species, while in others the large difference in size between mouse and human leads to different outcomes. A therapy that can successfully promote regeneration of a few mgs of murine skeletal muscle may be ineffective in the human where the muscle mass is several thousand folds higher. Although there is no substitute for the human to determine the success and possible side effects of any therapy, it is advisable that once a therapeutic approach has proven successful in the mouse, to test it in a large animal model with a tissue/organ biology and physiology as similar to the human as possible. The pig, because of its size, rapid growth rate, well known physiology and availability, has proven a very useful and frequently used preclinical large animal model for many pathologies, particularly those involving tissue regeneration. The main drawback is the absence of well characterized diseases of genetic origin and the still prohibitive costs of syngeneic strains that make cell and tissue transplantation dependent on immunosuppressive treatment with all the confounding factors that it implies. For this reasons, we have focusied our work on (i) isolating and characterizing porcine muscle stem cells (ii) analysing their response to growth factors and cytokines in vitro (iii) testing therapeutics approaches outlined in the previous projects on skeletal muscles regeneration models already setup.

Project Results:
The work performed since the beginning of the project covers all the areas mentioned above. We have initiated the development of several therapeutics that act on stem cells activation. These therapeutics have highest impact on opportunity to meet the challenges in regenerative medicine market.
Figure 1: ENDOSTEM ratioal and identified mechanisms

We have carried out preclinical studies on selected compounds with the aim of providing ground for their testing in clinical trials to assess their suitability as therapeutics. Of the various approaches, three have now reached clinical testing.
Endostem regroups clinical trials using therapeutics of 4 different pharmacological approaches; several clinical trials are scheduled or have already started in the last years:
• 2010: Phase IIa Clinical trial Using Nitric Oxide (NO) + NSAID as therapeutic agent
• 2013: Phase I studies using NO-NSAID combination
• May 2013: Phase Ib/2a Study of HDACi (Givinostat) in Duchenne Muscular Dystrophy (Should be completed August 2015)
• December 2014: Phase 1 pharmacokinetic, tolerability and feasibility study with omigapil in patients with Congenital Muscular dystrophy - CMD (Should be completed by September 2016)

Two other compounds, a soluble for Cripto and a superagonist of HMGB1 are in advanced preclinical development and emerged directly from the ENDOSTEM consortium.
We have investigated the molecular mechanisms that will allow us to understand the control of stem cell activation and differentiation. First results have been produced and are leading to promising new developments that are involved in the activation and mobilization of endogenous stem cell. For example we have identified several compounds (peptides or miRNA) that improve stem cell migration and recruitment during regenration. Below, we describe the work achieved since the beginning of the project focusing on clinical trials and therapeutics development.

4.1.3.1. Clinical trials
4.1.3.1.1. NO-NSAID trial
In 2010, we finalised a phase IIa study on 71 patients (36 of which with placebo) suffering from Duchenne, Becker and Limb Girdle Dystrophy. The aim of this open pilot study was to establish the profile of safety, tolerability and clinical response of NO and ibubrofen drug combination in a cohort of adult patients affected by muscular dystrophies. The study included 71 patients, of whom 35 treated with the drug combination for 12 months (10 with Duchenne Muscular Dystrophy, DMD, 7 with Becker Muscular Dystrophy, BMD, and 18 with Limb-girdle Muscular Dystrophy, LGMD) and 36 untreated (11 with DMD, 9 with BMD and 16 with LGMD). ISDN (isosorbide dinitrate) and ibuprofen are approved for use in humans with a good safety profile: we decided to test the effect of this combination in a pilot open study in dystrophic patients. The study was co-sponsored by the E. Medea Scientific Institute, the European Commission 7th framework programme and Parent Project Onlus. In the study we evaluated the safety and tolerability of the drug combination in a cohort of adult patients (Duchenne, Becker and Limb-girdle muscular dystrophy). The drug combination demonstrated a good profile of safety and tolerability, with only few and transient side effects. Additionally, we assessed some exploratory outcomes measures: left ventricle ejection and shortening fractions, forced vital capacity, forced expiratory volume and peak expiratory flow. We evidenced a trend towards amelioration although without statistical significance, as expected because the study had not been powered to investigate efficacy.
When we analysed the MFM scale (Motor Function Measure), a statistically significant difference was observed in the D1 subscale (standing-position change); in particular for LGMD (Limb girdle muscular dystrophy) patients values were 17.3 ± 9.1 and 32 ± 8.6 (p= 0.030) after 12 months of treatment. We conclude that systemic administration of ibuprofen and ISDN provides an adequate safety margin for clinical studies aimed at assessing efficacy and will be thus further developed in clinical trials specifically designed and powered to assess efficacy. Based on the results of this study we submitted for evaluations to the TACT committee network (TREAT-NMD Advisory Committee for therapeutics) our proposal for “A pharmacological treatment for muscular dystrophy combining NO-releasing and non steroidal anti-inflammatory drugs”. The proposal was reviewed in February 2010. The proposal was favourably judged but the committee also suggested further clinical studies to optimise dosing of the drug combination prior to initiating a phase II trial. Accordingly we designed two phase I studies in healthy volunteers as a step forwards the phase II clinical trial (ISOFEN1 and ISOFEN2). The two studies have been funded by Parent Project Italia Onlus.

4.1.3.1.1.1. ISOFEN 1 and ISOFEN 2: Study design/objective
ISOFEN1: to compare the pharmacokinetics plasma profile of ISDN 20 mg/day, ibuprofen 200mg/day and the combination of ISDN plus ibuprofen (20 mg/day + 200mg/day) given in single dose. In the study we compared the area under the time-concentration curve (AUC 0-12) of ibuprofen and ISDN when given concomitantly with AUCs measured in subjects given ISDN alone and ibuprofen alone, to describe pharmacokinetics parameters of the three treatments. We also evaluated the tolerability and safety of trial treatments given in a single dose. The study was carried out in 12 healthy volunteers. After the screening phase, subject who met inclusion and exclusion criteria were randomized to one of 3 sequences group. Trial treatments, i.e. ISDN 20 mg/day, ibuprofen 200mg/day and the combination of ISDN plus ibuprofen (20 mg/day + 200mg/day) were administered according the sequence randomization scheme with a wash out (wo) of at least 3 days between Periods.

ISOFEN2: To select the maximum tolerated dose of ISDN in combination with ibuprofen. In particular that the combination of ibuprofen 200 mg with ISDN up to 80 mg/day is well tolerated by at least the 80% of the subjects. Subjects started drugs intake at the lowest doses. At visits 3, 4 and 5 ISDN was up-titrated in subjects with a diastolic blood pressure (DBP) > 60 mmHg.

4.1.3.1.1.2. ISOFEN 1 and ISOFEN 2: Results, safey and tolerability study
ISOFEN 1: All enrolled subjects completed the study. Safety assessments, including vital signs, cardiac and respiratory evaluations showed no differences between the beginning and the end of the study. Likewise, no significant changes were observed in the haematological parameters tested, including red cells and platelets count, hepatic transaminases, electrolytes and glucose. Drug-related side effects were mild and transient: all subjects experienced headache, remitting spontaneously in 0.5-1 h, a known side effect of ISDN; one subject experiences transient nausea most likely related to the use of ibuprofen. No other drug related effects were observed. We conclude that the drug combination is safe and well tolerated.

ISOFEN2: All enrolled subjects completed the study. 94.7% of the subjects (18 subjects) tolerated the treatment well and completed the study at the highest dose of ISDN (80 mg/day). Only one subject after visit 5 (last week of treatment, when ISDN was up titrated to 80 mg as per protocol) decided to reduce the dosage of ISDN to 60 mg.
Safety assessments, including vital signs, cardiac and respiratory evaluations showed no differences between the beginning and the end of the study. Likewise, no significant changes were observed in the haematological parameters tested, including red cells and platelets count, hepatic transaminases, electrolytes and glucose. Drug-related side effects were mild and transient: all subjects experienced headache, remitting spontaneously in 0.5-1 h; regarding the onset of the headache, it has been examined whether there were differences about its repeatability.
In 11 cases headache presented only once after drug assumption. In 17 cases it occurred every time the drug was assumed. These recurrent episodes gradually decreased during the treatment period (8 cases at the beginning of the study, 1 case at the end of treatment). This tolerance effect to headache was also expected a it is commonly reported with ISDN. No other drug related effects were observed. We conclude that the drug combination is safe and well tolerated.

4.1.3.1.1.3. Foreground: Phase II study of the combination of iburpfen and isosorbide dinitrate in patients affected by Duchenne Muscular Dystrophy (ISOFEN 3)

1. Our hypothesis is that the combination of NO donation plus non steroidal anti-inflammatory activity obtained by combining the NO donor isosorbide dinitrate (ISDN) and the non steroidal anti-inflammatory agent (NSAID) ibuprofen has efficacy in slowing disease progression in Muscular Dystrophy.
2. Substantiation for the hypothesis
3. The evidence on the role of NO in muscle physiology, myogenesis and repair;
4. The efficacy of the combination of ISDN and ibuprofen as assessed in preclinical studies we carried out from 2006 onwards, detailed in the “pilot studies” section above;
5. Signs of amelioration in preliminary measures of efficacy in a 12-month clinical study we carried out in dystrophic patients with ISDN and ibuprofen detailed in the “pilot clinical study” section above. Importantly, this study demonstrated the safety and good tolerability profile of the drug combination;
6. The pharmacokinetic profile of the drugs proposed for the study, including the maximum tolerated dose, obtained in two phase I clinical studies detailed in the “studies in healthy volunteers” section above.

Therefore, based on this rational, this protocol describes the ISOFEN3 trial, a double blind, randomized, placebo-controlled phase II clinical study to assess the efficacy of the combination of the nitric oxide-releasing drug ISDN and the non steroidal anti-inflammatory drug ibuprofen in slowing the progression of Duchenne muscular dystrophy in 10-17 year old non-ambulant boys. In addition, safety and tolerability in terms of number of adverse events will be assessed.

4.1.3.1.2. Omigapil Trial
Omigapil, a small anti-apoptotic molecule originally developed by Novartis for the treatment of Parkinson’s Disease and Amyotrophic Lateral Sclerosis, acts by interacting with glyceraldeyde 3-phosphate dehydrogenase (GAPDH) and protecting from GAPDH-mediated apoptosis; furthermore, omigapil and related molecules preserve mitochondrial membrane from the GAPDH-mediated pro-apoptotic transmembrane potential loss, reduce p-53 dependent cell death and increase levels of the anti-apoptotic molecule Bcl-2 in PC12 cells forced to apoptosis by serum withdrawal.

4.1.3.1.2.1. Rationale for a study on MDC1A and UCMD/BM patients
The most prominent CMD forms result from deficiency in laminin-α2, (MDC1A), or from deficiency in collagen VI in the case of both both UCMD (Ullrich congenital muscular dystrophy) and BM (Bethlem Myopathie). . There is a well-established mouse model, the homozygous dyW mouse, which reiterates several key aspects of MDC1A pathology including kyphosis, reduced weight gain, impaired locomotion, limited life span and dystrophic features in muscle histology including apoptosis. Apoptosis in muscle cells is a general feature in animal models of CMD. The muscle of MDC1A patients is characterised by an early inflammatory process, marked variation in muscle fibre size, extensive fibrosis and proliferation of adipose tissue. Furthermore, the basement membrane which surrounds the muscle fibres and lacks laminin- α2 is disrupted, resulting in the detachment and subsequent apoptosis of the muscle fibres.
The collagen VI deficient mouse (Col6a1-/-) is an animal model of BM and UCMD showing histological signs of myopathy (variation in fibre diameter) and a latent mitochondrial defect, causing muscle apoptosis. Latent mitochondrial dysfunction is observed in myoblasts from UCMD patients that matched an increased occurrence of spontaneous apoptosis.

4.1.3.1.2.2. CALLISTO: study design
The phase 1 study has been registered on March 2013 and is titled officially CALLISTO (Congenital Muscular Dystrophy, Ascending Multiple Dose Cohort Study anaLyzing Pharmacokinetics at three dose Levels in Infants and Adolescents and assessing Safety and Tolerability of Omigapil)
• Primary Endpoint: Pharmacokinetic profile of omigapil and main metabolites (Cmax, Cmin, Tmax, T1/2, AUC0-8h)
• Secondary Endpoint: Safety and tolerability of omigapil established through the collection of Adverse Events (AEs) and Serious Adverse Events (SAEs), standard lab parameters and ECG assessments
• Tertiary Endpoint: Feasibility of assessing disease-relevant clinical outcome measures.

4.1.3.1.2.3. Foreground: CALLISTO 2
Santhera obtained the orphan drug designation for omigapil from EMA (European Medicines Agency) and FDA (Food and Drug Agency) in 2009 and completed the non-clinical development required for the use of the drug in paediatric patients by 2010, with the support of a grant from the Association Française contre les Myopathies. The preparation of the further clinical development started in 2010 with the collaboration of the patient advocacy organization CureCMD and with NINDS (National Institute of Neurological Disorders and Stroke) clinical experts in advancing translational research into the clinic. The participation in the EU-funded program ENDOSTEM from September 2011 enabled Santhera to progress on the clinical development of the first therapeutic approach for this devastating group of diseases. No treatment is presently available for children affected by MDC1A and Ulrich’s/Bethlem Myopathies. There is a high unmet medical need for neglected forms of congenital myopathy. The CALLISTO study results, expected in 2016, will enable to evaluate the tolerability and pharmacokinetic profiles of omigapil in children and investigate the feasibility of efficacy assessments for future studies:
1. Conduct Phase I study in CMD patients CALLISTO 2. Evaluate results from Phase I study
2. Further clinical development plan
• Define dose for Phase II/III
• Define suitable clinical endpoints for Phase II/III
• Development of Phase II/III / market formulation
• Discussion with Regulatory Authorities
3. Phase II/III efficacy study in CMD patients
4. In parallel to Phase II/III study:
• Carcinogenicity studies
• Clinical Pharmacology studies (tbd)
• Technical development: API and formulation registration / validation batches
5. NDA /MAA filing following demonstration of efficacy & safety

4.1.3.1.3 Givinostat Clinical Trial
Givinostat is a Histone Deacetylase Inhibitor (HDACi) and it was granted Orphan Drug designation both in EU and USA for the treatment of DMD.

4.1.3.1.3.1. Rationale for a study on DMD PATIENTS
Italfarmaco (ITF) is developing Givinostat for the treatment of Duchenne Muscular Dystrophy (DMD). Following the demonstration of a dose- and concentration- dependent increase in muscle and reductions in inflammation, fatty replacement and fibrosis in a DMD mouse model, ITF designed a Proof of Concept study to demonstrate that Givinostat could exert similar effects also in DMD children.

4.1.3.1.3.2. Givinostat clinical trial: “A two part study to assess safety and tolerability, pharmacokinetics, effects on histology and on different clinical parameters of Givinostat in ambulant children with Duchenne Muscular Dystrophy “

In this study, after an initial dose escalation phase lasting approximately 2 months, 19 children 7-10yrs of age, on at least 6 months of stable steroids and walking at least 250 meters at baseline, were treated for 12 additional months at the selected dose (37.5 mg b.i.d. reduced to 25 mg b.i.d. in 12 children to maintain platelet counts within the normal range). Primary study objective was the evaluation of the histologic effects of Givinostat comparing baseline and end of treatment muscle biopsies (brachial biceps). Functional assessments (6 Minute Walk Test, 6MWT; North Star Ambulatory Assessment, NSAA) were secondary endpoints as well as safety and tolerability.
Once all 20 children enrolled during the Part 1 of the study have been treated for at least 2 weeks, the review team will determine the recommended dose (RD) to be used in Part 2 based on the safety and tolerability profile observed and on the pharmacokinetic (PK) analyses. All the children enrolled will switch to the RD level, which will be administered for the subsequent 12 months of the study (Part 2).
The additional children (if any) will be enrolled during Part 2 of the study and will receive the RD of Givinostat for 12 months. The primary objective is to measure the change in the value of muscle fiber atrophy percentage comparing the histology biopsies before and after 12 months of treatment with Givinostat.

Part 1 results summary:
• Maximum Tolerated dose has been found
o 25mg bi-daily (bid): tolerated;
o 50mg bid: not tolerated;
 Dose Limiting Toxicity is Thrombocytopenia
o 37,5mg bid: tolerated
• The Recommended Dose for Part 2 of the study (i.e. 37,5 mg BID)
• Safety profile – additional information: some mild diarrhea events were observed

Part 2 results summary
• 19 children started GIVINOSTAT at 37.5 mg bid on July 2013 .
• After 2 months of treatment, the dose was reduced at 25 mg bid, in subjects for which the platelet count was ≤150 x 109/L :
o 12 children are treated at the dose of 25mg bid
o 7 children are currently in treatment with 37,5 mg bid
• Some diarrhea events are observed but they are mild
• The children will be treated till July 2014 (48 weeks of treatment)

4.1.3.1.3.3. Foreground extension of Givinostat trial
Children are continuing treatment as the study has been extended for additional 12 months to evaluate long-term tolerability.
Safety and efficacy results relevant to the first part (dose finding phase) and second part (proof of concept phase) will be shown during the annual Parent Project Conference (on 21 February 2015).

The study is being conducted in 4 centres in Italy:
• Dipartimento di Scienze Neurologiche, I.R.C.C.S. Fondazione Ca’Granda, Ospedale Maggiore Policlinico, Milano (Principal Investigator: Prof. Giacomo Pietro Comi);
• UO di Malattie Neuromuscolari e Neurodegenerative dell’Ospedale Pediatrico Bambino Gesù, Roma (Principal Investigator: Prof. Enrico Bertini);
• UO di Neuropsichiatria Infantile, Policlinico Gemelli, Roma (Principal Investigator: Prof. Eugenio Mercuri);
• UOC Neurologia e Malattie Neuromuscolari, Policlinico Universitario di Messina (Principal Investigator: Prof. Giuseppe Vita)

4.1.3.2. Targeting Muscle Stem Cells
Understanding the mechanism underlying muscle stem cells activation allowed us the identification of molecules that can be used to activate muscle regeneration through endogenous stem cell cells mobilization. During the last five years significant progress has been achieved and candidate molecules have been identified.

4.1.3.2.1. Dissceting muscle stem cells lineage and activation mechanism
1. We have shown that growth arrest and differentiation are coordinated via the Notch signaling pathway during skeletal muscle regeneration.

2. We have identified candidate genes to be targeted in order to promote efficient skeletal muscle regeneration and shown that SoxF genes are necessary for the maintenance of satellite cell quiescence.

3. Several partners have shown the existence of different stem cells lineages that co-exist in skeletal muscle. Most studies show the importance of the signaling and the crosstalk between these lineages. These data indicate that PW1 interstitial cells (PICs) and fibroadipogenic progenitors (FAPs) provide a novel source of endogenous, pharmacologically inducible, population of intramuscular cells that can be exploited to regenerate dystrophic muscles and prevent deleterious events, such as fibro-adipose infiltration of muscles that complicate DMD progression.

4. On the matter of lineage interactions and looking for new therapeutical targets, we have shown that autocrine IGF-1 is an M2 polarizing stimulus and that administration of IGF-1Ea or blocking of IGF-1 can used to modulate the macrophage population with a significant resulting impact on muscle repair.
5. We analyzed the mechanism of action of nitric oxide in the activation of myogenesis and we have found that NO actions appear coordinated to increase SC number per fibre.

6. In the aim of identifying new therapeutic targets, we characterized 2 miRNAs that have been previously poorly described with functional relevance in skeletal muscle. Both miRNAs are highly upregulated in different muscle disease models thereby representing attractive targets for further investigations.
Muscle satellite cells are the obvious cellular target, however little was known about:
• Control of satellite cell activation
• Entry into cell cycle quiescence (Go)
• Cell fate choices mechanisms.
We have pursued during the last five years the fundamental characterization of the actual target cells in the muscle repair process in order to intelligently design future therapeutic approaches. A significant progress has been achieved in the comprehension of the molecular mechanisms and new targets have been identified thanks to a large scale profiling of progenitors and postnatal stem cells from embryonic day 8.5 to old mice of 24 months.

Figure 2: Large scale profiling of molecular mechanism involved in myogensis from developmental age of E8.5 to 24 months old mice

In addition to the characterisation of satellite cells, other muscle resident cell populations such as the PICs (PW1 expressing Interstitial Cells) or ‘circulating’ stem cells have been shown to participate fundamentally in muscle regeneration. We have shown that there is an essential crosstalk between PICs and satellite cells involved in muscle growth as well as in cell fate choices during muscle regeneration.
Figure 3: Crosstalk between Pics and satellite cells involved in cell growth and cell fate

4.1.3.2.2. Foreground: development of newly identified compounds
We have identified a novel relevant pharmaceutical candidate, sCripto, regulating skeletal muscle regeneration and stem cell regulation.
Figure 4: Cripto involvment in skeletal muscle regeneration

4.1.3.3. Targeting Vascular Associated Stem Cells
4.1.3.3.1. VAPs: new targets for muscular regeneration
Understanding the pathways in which endogenous vascular progenitors contribute to muscle regeneration was one of the most innovative approach of ENDOSTEM consortium. We analyzed the contribution of VAPs (Vascular associated progenitors) to muscle regeneration by (i) the induction of new vessels and therefore by increasing tissue perfusion and oxygenation; (ii) through differentiation in new muscle fibers. We have studied the best conditions to improve both processes aimed to bring new adjuvant therapies to muscle regeneration. During the last five years we have reached all the objectives and we have identified strategies to increase vascular progenitors mobilization and homing that we have shown as beneficial for muscle neo-vascularization and regeneration.

We have isolated VAPs and identified PW1 as a suitable marker for a sub-population of endothelial progenitors and its expression determines mesoangioblasts (MABs) stem cell competence.
We have shown that involvement of different pathways in the mobilization of VAPs (Vascular associated progenitors), including wnt signaling, VE-Cadherin signaling, JAM-A, SDF-1 and HMGB1.

This understanding of vascular stem cells homing allowed our consortium to identify and to define pharmacological treatments for muscle repair targeting different levels of muscular regeneration:
Vascular stem cells mobilization: We have tested several miRNAs to improve homing of bone marrow-derived cells. We showed that miR-34a inhibition improved homing functions in vitro and in vivo. However, several other miRNAs, such as miR-92a (which is expected to target SDF-1) did not affect bone marrow-derived cell functions. We are currently testing miR-210 and let-7 as putative targets.

Skeletal Muscle angiogenesis: We generated a HMGB1 (High–mobility group box 1) mutant form (3S) resistant to oxidation. Interestingly, this mutant behaves as a superagonist for cell recruitment and it fails to induce the expression of proinflammatory cytokine and chemokine by immune cells. Data demonstrated that HMGB1 has beneficial effects in skeletal muscle regeneration after acute injury by increasing the number of healthy fibres and of satellite cells. Moreover, HMGB1 acts directly on primary myoblasts by inducing their migration and their fusion to form large myotubes. Remarkably, 3S-HMGB1 behaves as a superagonist of HMGB1 in vivo, suggesting that it is a very promising candidate for muscle repair therapies. The superagonist HMGB1 has been shown to increase the expression of proangiogenic markers correlated to an increase in muscle vascularization and myofibers formation.

Vessel Permeability: We showed that JAM-A is involved in the formation of the new skeletal myofibers on muscle vascularization and showed that the inhibition of JAM-A promotes the formation of new vessels. Therefore, JAM-A became a new target for improving vascular cell therapy for muscular dystrophy. Using JAM-A antibody that is involved in vessels permeability, we have shown that transmigration of mesoangioblats (VAPs) is facilitated, as the junctions are less tight, favoring mesoangioblats engraftment. Very recently we have also shown that PW1 regulates JAM-A directly by binding to its promoter, consequently, it can be a new target for modulating vascular stem cells competence. Finally using NO donors we have shown that NO enhance stem cell homing through vasodilatation and permeability and may act at different levels of muscle régénération pathways.

Figure 5: Nitric Oxide has protective role in skeletal muscle tissue biology

4.1.3.3.2. Foreground: Development of newly idetified compounds for muscle dystrophy
Several compounds have been identified and their mechanism of action carcterized, among them HMGB1 superagonist. The development of this compound is ongoing in HMGB biotech in the aim of reaching clinical trials in near future. The other promising targeting will be miRNA identified by the team of Stefanie Dimmeler, that can be used as an alterantive or a complementary approach to stem cells transplantation.

4.1.3.4. Targeting immunological, fibrotic and inflammatory mechanisms
4.1.3.4.1. Macrophages polairzation and muscle regeneration
Tissue damage and cell necrosis initiate the rapid and sequential invasion of muscle by inflammatory cells, such as leukocytes and macrophages. We have shown that stem cells are also affected in this process. Recruited leucocytes play crucial, although apparently contrasting, roles: they are required for activation and differentiation of precursor cells and for matrix assembly. At the same time they actively amplify the damage. Combining in vivo and in vitro data we have shown that human skeletal muscle regeneration is sequentially associated with several macrophages (MP) subsets. Furthermore, we provided evidences of new functions of MPs in the coordinated regulation of MPC fate during muscle repair. These results strengthen the emerging concept that inflammation regulates stem cell homeostasis. The characterization of macrophages biomarkers during muscle regeneration allowed us to define the spectrum of macrophage polarization defined as an equilibrium that can be altered in dystrophic conditions and consequently lead to fibrosis in stead of muscle regeneration and revascularization.

Figure 6: Macrophage polarization spectrum

We have also studied and identified the mechanisms involved in macrophage phenotypic transitions. The results of these studies have uncovered a novel regulatory mechanism involving the appropriate balance of p38, MKP-1, miR-21, and AKT activities, that may have implications in chronic inflammatory degenerative diseases. To get further insight into the mechanism controlling inflammation-mediated fibrosis progression in dystrophic context; PM is analyzing the role of matrix-associated fibrinogen. For this purpose PM performed a therapeutic treatment with the FIGGAMMA377-395 peptide (blocking ALPHAMBETA2 integrin receptor binding and macrophage activation), and showed that in vivo treatment with the Fibγ377-395 peptide attenuates the severity of muscular dystrophy via selective interference with macrophage activation in necrotic muscle without undesirable deleterious hemorrhagic effects. These finding were confirmed by the generation of a new tool, a new mouse model where a macrophage-specific promoter, the promoter of CD163, drives the suicide gene human Diphteria toxin receptor (hDTR), to selectively kill polarized CD163high macrophages. Using this newly generated tool we demonstrated that infiltrating macrophages affect the fate of endothelial derived progenitors and inhibits their Endothelial to Mesenchymal transition (EndoMT) during muscle regeneration. Macrophage depletion indeed leads to EndoMT, fibroblast expansion and fibrosis of the muscle.
Figure 7: Macrophage polarization defects lead to fibrosis and heterotopic ossification

4.1.3.4.2. Foreground: innovative therapeutic targets for muscle regeneration
4.1.3.4.2.1. Nitric Oxide donors
We showed that Nitric Oxide donation in dystrophic mice using the drug Molsidomine, modulates effectively, in a time dependent way, the characteristics of the inflammatory infiltrate within dystrophic muscles, enhancing its healing function. Initially molsidomine amplified macrophage recruitment, promoting a more efficient clearance of cell debris and a more effective regeneration of the tissue. At a later stage molsidomine decrease significantly the extent of the inflammatory infiltrate, whose persistence exacerbates muscle damage: of the remaining macrophages the majority displayed characteristics of transitional population, associated with a reduced fibrosis and increased preservation of the muscle tissue.

4.1.3.4.2.2. Chemokines and chemokine receptors (CCR) modulators
We have specifically addressed the implication of specific chemokines in tissue repair. For doing this, we analyzed the pathological phenotype study of 6 mouse lines deficient for CCR1, CCR2, CCR5, CX3CR1, CCL2/CX3CR1 and CXCR3. The results suggest that a complex set of CK/CKR axis orchestrate muscle regeneration:
• CX3CR1 deficiency improves muscle healing associated with an increased of both number and size of regenerating myofiber whereas CCL2 deficiency leads to severe muscle regeneration impairment.
• Additional parameters of muscle regeneration i.e. fat accumulation, muscle fibers calcification, fibrosis development and neo-vascularisation are improved in Cx3cr1 deficient mice but are worse in Ccl2 and Ccr2-deficient mice.
• Double deficient (Cx3cr1-/- Ccl2-/-) mice exhibited a better regeneration than Ccl2-/- mice with increased regenerating myofibers size and reduced calcification, adipocytosis, fibrosis and increased vascularization.
These results indicated that CX3CR1 deficiency improved muscle regeneration in Ccl2-/- mice and suggested that both CCR2 and CX3CR1 axis may exert different functions in muscle regeneration. This raised the possibility of therapeutic intervention by manipulating chemokine system. This year results of CC indicated that CX3CR1 deficiency improved muscle regeneration in CCL2-/- mice and suggested that both CCR2 and CX3CR1 axis may exert different functions in muscle regeneration. More specifically, antagonists and agonists of the CX3CR1 receptor have been identified. These antagonists and agonists can be used for treating an inflammatory disorder, an autoimmune disorder, a cardiovascular disease, a neurodegenerative disease, a graft versus host disease, a behavioral disorder, a cicatrisation disorder, a viral infection, cancer or pain.

4.1.3.5. Tissue Microenvironment & Pharmacological Approaches, Delivery and Mechanisms

4.1.3.5.1. Understanding mechanisms underlying stem cell reprogramming
We aimed in the understanding of the mechanisms underlying the global and focal changes in chromatin structure – the stem cell nuclear reprogramming – which is essential to devise pharmacological interventions that can manipulate expression of key genes to promote stem cell-based repair of injured or diseased organs and tissues.
We have identified and characterized several non-satellite myogenic progenitors as well as non-myogenic populations that support the muscle regenerative process (PW1-positive interstitial cells, PICs and FibroAdipogenic progenitors, FAPs. Comparative transcriptome analysis of resident muscle stem cells allowed us to define the fate potential of each population and to identify new pathways and therapeutics that could lead to a better regeneration.

Chronic myopathies exhibit a replacement of myofibers by fibrosis and fat concomitant with a decrease in resident muscle stem cells (satellite cells). Pharmacological interventions could also lead to changes in the muscle environment and the stem cells niche. One common feature in these conditions is the generation of novel external cues, which elicit intracellular signalling pathways that influence gene transcription. Understanding how these signals are interpreted at the chromatin level and converted into epigenetic modifications that regulate the expression of key genes is essential to devise specific therapeutic strategies toward selective manipulation of gene expression in muscle progenitors. To this end we have analyzed the epigenetic profiles of pathologic and healthy muscles in mice. We identified genes that influence the fate of stem cells and that can be targeted in novel therapeutical approaches. We have shown that environmental cues from the stem cell niche control the selection of specific subunit variants of the switch/sucrose non-fermentable (SWI/SNF) chromatin-modifying complex, creating a combinatorial code that dictates whether cells adopt myogenic versus non-myogenic cell fates.

Stepwise recruitment of SWI/SNF to MyoD-target genes by pre-assembled BAF60c–MyoD complex. In undifferentiated myoblasts, MyoD and BAF60c bind the myogenin promoter in the absence of Brg1-based SWI/SNF core complex. SWI/SNF core is recruited to the promoter by differentiation-activated p38 kinase α/β, through phosphorylation of BAF60c on threonine 229, thereby promoting chromatin remodelling and assembly of the transcriptosome for initiation of transcription.
(Forcales et al, 2012)

Polymer nanoparticle and micelle formulations can confer new properties to existing molecules, in order to improve targeting and prolonged delivery and to reduce of off-target effects. Previous efforts have been directed to nitric oxide (NO) delivery. In this project, we have turned our attention to carbon monoxide (CO)2, 6-tioguanine3 and ibuprofen4.

4.1.3.5.2. Foreground: Clinical trials and innovative compounds developement
In collaboration with Italfarmaco, we performed preclinical studies to define appropriate therapeutic dosage and paradigm for Givinostat which is a HDAC inhibitor. The compound is now in phase I/II clinical trial.
We utilized also other approaches, such as high throughput screening in order to identify other compounds that modulate the epigenetic profile and the expression of key genes in individual populations of endogenous stem cells. By doing this we identified some microRNA that are directs chromatin remodelling of genes implicated in opposite activities – fibro-adipogenesis vs myogenesis. As such, this screening paves the way for further screening strategies toward identifying compounds that modulate microRNA expression in FAPs.

Finally, thanks to our understanding of epigenetic modulators of stem cell fate, we developed an ex vivo model that allows to screen the effect of compound libraries on endogenous muscle stem cell.

4.1.3.6. Developing large animal models for preclinical development
It is essential for pre-clinical testing of therapies for dysfunctional muscle to be carried out in a model, which is more similar than the available rodent models in tissue biology, size and physiology to the human. The porcine model represent a good compromise to optimize analysis, due to its size, rapid growth rate, well known physiology and availability, has proven a very useful and frequently used pre-clinical large animal model for many pathologies.
One challenge was to identify and to characterize the porcine muscle stem cells. The second challenge was to set up the optimized conditions to maintain muscle stem cell growth in genetic and phenotypic stability and to generate large quantities of pig skeletal muscle-derived stem-progenitor cells. We have succeeded to meet these both challenges.
Considering the key role of soluble factors in mediating intercellular interactions through autocrine and paracrine signalling, the secretome can be considered as a clinically relevant source for regenerative therapies. As such, the identification of growth factors and cytokines, which mediate the proliferation and commitment of skeletal muscle stem cells, is a crucial step toward the discovery of new molecular targets for clinical therapy. This last year we have identified a panel of growth factors, which are involved in cell cycle and myogenic differentiation.

4.1.3.6.1. Foreground: a new validated model for preclinical development for muscular dystrphies treatement
We have demonstrated the relevancy of the porcine model, not only for skeletal muscle regeneration but also for cardiac muscle.
Particularly we had identified and characterized muscle stem cells using tools that have been identified from other Endostem partners. We have also set up two preclinical models to assess efficacy of candidate’s compounds and finally we tested two growth factors.

Potential Impact:
The proposed projects capitalize on combined expertise in different areas of regenerative medicine. The proposal involves collaborative interactions that allow us to merge our unique and complementary expertise in the field.

Impact on science
Presently, there is a focus on the therapeutic use of engrafted stem cells to treat degenerative diseases or aging; however advancing our understanding of the basic biology of stem cell activation best complements such efforts. Regardless of the potential success of engrafted stem cells, such therapies will be very costly and will require tailoring for each patient given the current state-of-the-art. Approaches aimed at mobilizing endogenous stem cells become more plausible in light of a major shift in the field of adult stem cell biology that has provided increasing evidence that pluripotent stem cells with regenerative potential are present in adult tissues contrary to the generally accepted view just 5 to 10 years ago. While many tissues possess limited regenerative potential, the capacity for regeneration declines with age and chronic disease. In addition, stem cell recruitment in response to injury or disease often produces inappropriate re-patterning of the tissue culminating in scar tissue formation (fibrosis), inadequate revascularisation, or chronic inflammatory disorders.

We are developing new strategies to activate and mobilize tissue-associated endogenous stem cells as a tool for efficient tissue repair and as an alternative/complementary approach to stem cell transplantation. The starting point for our proposal is the combined research effort by the partner groups focused upon candidate agents that target muscle and muscle vasculature progenitor cells as well as preventing tissue damage to optimize endogenous stem cell function.

The last five years were flourishing in the identification of new target genes and new bioactive molecules that act on the muscular and vascular systems as well as the interactions between the several stem cell subpopulations present in the muscle tissue. Moreover we have initiated three clinical trials using epigenetic modifiers and chemical entities.

Impact on society
Degenerative and age related diseases create a life-altering experience for the person with injury, for their partner, parents, siblings, and children. The subsequent diminishment of body functions associated with the diseases can cause depression and loss of self-esteem. It has been considered essential, based on European policy consistent with human rights principles, that people with disabilities should be treated with dignity, encouraged to have independence, be given equality of opportunity, encouraged to have an active participation, a full citizenship and a high quality of life. Given the diversity of degenerative diseases indicated above, pathological manifestation can occur at any age: either as a child, during an individual's most productive years, or as an aged person. The trauma frequently results in morbidity, and as a result, patients typically require continuous physical and medical care depending on the disease, severity of manifestation, degree of disability, and location of injury.

Economic impact
The prevalence of degenerative diseases is on the rise because aging population is increasing and this has created the need for biomaterials. Over the past 50 years, average life expectancy at birth has increased globally by over 20 years, from 46.5 years in 1950-55 to 65.2 years in 2002. Today there are 600 million people in the world aged 60 years or over, and this will double by 2025 and reach 2 billion by 2050. While degenerative diseases are not the exclusive domain of the aged, they do impact this sector of society the highest with subsequent increased social and economic burdens on the health care systems on which they depend.

The direct healthcare costs of organ replacement are about € 240 billion globally (about 8 percent of global healthcare spending) arising from therapies that keep people alive (such as kidney dialysis), implanted replacement devices, and organ transplants. With a € 240 billion global industry already built on first generation tissue and organ therapy products and substitutes, regenerative medicine has a potential to exceed € 600 billion by 2030.

EndoStem and its partners have demonstrated this year a proof of principle through the interaction and integration of the fundamental, pre-clinical and clinical partners that have expertise in different fields such as muscular, vascular, epigenetic, immunologic fields.

By providing new and efficient therapeutics, Endostem will decrease certain social burdens while increasing the economic potential of Europe’s leading innovators in this exciting field.