Intra‐arterial transplantation of HLA ‐matched donor mesoangioblasts in Duchenne muscular dystrophy
2015; Springer Nature; Volume: 7; Issue: 12 Linguagem: Inglês
10.15252/emmm.201505636
ISSN1757-4684
AutoresGiulio Cossu, Stefano C. Previtali, Sara Napolitano, Maria Pia Cicalese, Francesco Saverio Tedesco, Francesca Nicastro, Maddalena Noviello, Urmas Roostalu, Maria Grazia Natali Sora, Marina Scarlato, Maurizio De Pellegrin, Claudia Godi, Serena Giuliani, Francesca Ciotti, Rossana Tonlorenzi, Isabella Lorenzetti, Cristina Rivellini, Sara Benedetti, Roberto Gatti, Sarah Marktel, Benedetta Mazzi, Andrea Tettamanti, Martina Ragazzi, Maria Adele Imro, Giuseppina Marano, Alessandro Ambrosi, Rossana Fiori, Maria Pia Sormani, Chiara Bonini, Massimo Venturini, Letterio S. Politi, Yvan Torrente, Fabio Ciceri,
Tópico(s)Cytomegalovirus and herpesvirus research
ResumoResearch Article5 November 2015Open Access Source Data Intra-arterial transplantation of HLA-matched donor mesoangioblasts in Duchenne muscular dystrophy Giulio Cossu Corresponding Author Giulio Cossu Institute of Inflammation and Repair, University of Manchester, Manchester, UK Search for more papers by this author Stefano C Previtali Corresponding Author Stefano C Previtali Institute of Experimental Neurology (InSpe), Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy Department of Neurology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Sara Napolitano Sara Napolitano HSR/TIGET Pediatric Clinical Research Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Hematology and BMT Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Maria Pia Cicalese Maria Pia Cicalese HSR/TIGET Pediatric Clinical Research Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Hematology and BMT Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Francesco Saverio Tedesco Francesco Saverio Tedesco Department of Cell and Developmental Biology, University College London, London, UK Search for more papers by this author Francesca Nicastro Francesca Nicastro Laboratory of Analysis and Rehabilitation of Motor Function, Division of Neurosciences, IRCCS San Raffaele Scientific Institute, Milan, Italy Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy Search for more papers by this author Maddalena Noviello Maddalena Noviello Experimental Hematology Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Urmas Roostalu Urmas Roostalu Institute of Inflammation and Repair, University of Manchester, Manchester, UK Search for more papers by this author Maria Grazia Natali Sora Maria Grazia Natali Sora Department of Neurology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Marina Scarlato Marina Scarlato Department of Neurology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Maurizio De Pellegrin Maurizio De Pellegrin Unit of Orthopaedics, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Claudia Godi Claudia Godi Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy Neuroradiology Department and Neuroradiology Research Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Serena Giuliani Serena Giuliani Hematology and BMT Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Francesca Ciotti Francesca Ciotti Hematology and BMT Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Rossana Tonlorenzi Rossana Tonlorenzi Institute of Experimental Neurology (InSpe), Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Isabella Lorenzetti Isabella Lorenzetti Institute of Experimental Neurology (InSpe), Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Cristina Rivellini Cristina Rivellini Institute of Experimental Neurology (InSpe), Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Sara Benedetti Sara Benedetti Department of Cell and Developmental Biology, University College London, London, UK Search for more papers by this author Roberto Gatti Roberto Gatti Laboratory of Analysis and Rehabilitation of Motor Function, Division of Neurosciences, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Sarah Marktel Sarah Marktel Hematology and BMT Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Benedetta Mazzi Benedetta Mazzi Immunogenetics Laboratory, Department of Immunohematology & Blood Transfusion, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Andrea Tettamanti Andrea Tettamanti Laboratory of Analysis and Rehabilitation of Motor Function, Division of Neurosciences, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Martina Ragazzi Martina Ragazzi Department of Cell and Developmental Biology, University College London, London, UK Search for more papers by this author Maria Adele Imro Maria Adele Imro MolMed S.p.A., Milan, Italy Search for more papers by this author Giuseppina Marano Giuseppina Marano MolMed S.p.A., Milan, Italy Search for more papers by this author Alessandro Ambrosi Alessandro Ambrosi University San Raffaele Vita e Salute, Milan, Italy Search for more papers by this author Rossana Fiori Rossana Fiori Unit of Anesthesiology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Maria Pia Sormani Maria Pia Sormani Department of Health Sciences, University of Genoa, Genoa, Italy Search for more papers by this author Chiara Bonini Chiara Bonini Experimental Hematology Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Massimo Venturini Massimo Venturini Department of Radiology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Letterio S Politi Letterio S Politi Neuroradiology Department and Neuroradiology Research Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Yvan Torrente Corresponding Author Yvan Torrente Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy Search for more papers by this author Fabio Ciceri Corresponding Author Fabio Ciceri HSR/TIGET Pediatric Clinical Research Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Giulio Cossu Corresponding Author Giulio Cossu Institute of Inflammation and Repair, University of Manchester, Manchester, UK Search for more papers by this author Stefano C Previtali Corresponding Author Stefano C Previtali Institute of Experimental Neurology (InSpe), Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy Department of Neurology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Sara Napolitano Sara Napolitano HSR/TIGET Pediatric Clinical Research Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Hematology and BMT Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Maria Pia Cicalese Maria Pia Cicalese HSR/TIGET Pediatric Clinical Research Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Hematology and BMT Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Francesco Saverio Tedesco Francesco Saverio Tedesco Department of Cell and Developmental Biology, University College London, London, UK Search for more papers by this author Francesca Nicastro Francesca Nicastro Laboratory of Analysis and Rehabilitation of Motor Function, Division of Neurosciences, IRCCS San Raffaele Scientific Institute, Milan, Italy Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy Search for more papers by this author Maddalena Noviello Maddalena Noviello Experimental Hematology Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Urmas Roostalu Urmas Roostalu Institute of Inflammation and Repair, University of Manchester, Manchester, UK Search for more papers by this author Maria Grazia Natali Sora Maria Grazia Natali Sora Department of Neurology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Marina Scarlato Marina Scarlato Department of Neurology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Maurizio De Pellegrin Maurizio De Pellegrin Unit of Orthopaedics, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Claudia Godi Claudia Godi Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy Neuroradiology Department and Neuroradiology Research Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Serena Giuliani Serena Giuliani Hematology and BMT Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Francesca Ciotti Francesca Ciotti Hematology and BMT Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Rossana Tonlorenzi Rossana Tonlorenzi Institute of Experimental Neurology (InSpe), Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Isabella Lorenzetti Isabella Lorenzetti Institute of Experimental Neurology (InSpe), Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Cristina Rivellini Cristina Rivellini Institute of Experimental Neurology (InSpe), Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Sara Benedetti Sara Benedetti Department of Cell and Developmental Biology, University College London, London, UK Search for more papers by this author Roberto Gatti Roberto Gatti Laboratory of Analysis and Rehabilitation of Motor Function, Division of Neurosciences, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Sarah Marktel Sarah Marktel Hematology and BMT Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Benedetta Mazzi Benedetta Mazzi Immunogenetics Laboratory, Department of Immunohematology & Blood Transfusion, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Andrea Tettamanti Andrea Tettamanti Laboratory of Analysis and Rehabilitation of Motor Function, Division of Neurosciences, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Martina Ragazzi Martina Ragazzi Department of Cell and Developmental Biology, University College London, London, UK Search for more papers by this author Maria Adele Imro Maria Adele Imro MolMed S.p.A., Milan, Italy Search for more papers by this author Giuseppina Marano Giuseppina Marano MolMed S.p.A., Milan, Italy Search for more papers by this author Alessandro Ambrosi Alessandro Ambrosi University San Raffaele Vita e Salute, Milan, Italy Search for more papers by this author Rossana Fiori Rossana Fiori Unit of Anesthesiology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Maria Pia Sormani Maria Pia Sormani Department of Health Sciences, University of Genoa, Genoa, Italy Search for more papers by this author Chiara Bonini Chiara Bonini Experimental Hematology Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Massimo Venturini Massimo Venturini Department of Radiology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Letterio S Politi Letterio S Politi Neuroradiology Department and Neuroradiology Research Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Yvan Torrente Corresponding Author Yvan Torrente Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy Search for more papers by this author Fabio Ciceri Corresponding Author Fabio Ciceri HSR/TIGET Pediatric Clinical Research Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Author Information Giulio Cossu 1, Stefano C Previtali 2,3, Sara Napolitano4,5, Maria Pia Cicalese4,5, Francesco Saverio Tedesco6, Francesca Nicastro7,8, Maddalena Noviello9, Urmas Roostalu1, Maria Grazia Natali Sora3, Marina Scarlato3, Maurizio De Pellegrin10, Claudia Godi8,11, Serena Giuliani5, Francesca Ciotti5, Rossana Tonlorenzi2, Isabella Lorenzetti2, Cristina Rivellini2, Sara Benedetti6, Roberto Gatti7, Sarah Marktel5, Benedetta Mazzi12, Andrea Tettamanti7, Martina Ragazzi6, Maria Adele Imro13, Giuseppina Marano13, Alessandro Ambrosi14, Rossana Fiori15, Maria Pia Sormani16, Chiara Bonini9, Massimo Venturini17, Letterio S Politi11, Yvan Torrente 8 and Fabio Ciceri 4 1Institute of Inflammation and Repair, University of Manchester, Manchester, UK 2Institute of Experimental Neurology (InSpe), Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy 3Department of Neurology, IRCCS San Raffaele Scientific Institute, Milan, Italy 4HSR/TIGET Pediatric Clinical Research Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy 5Hematology and BMT Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy 6Department of Cell and Developmental Biology, University College London, London, UK 7Laboratory of Analysis and Rehabilitation of Motor Function, Division of Neurosciences, IRCCS San Raffaele Scientific Institute, Milan, Italy 8Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy 9Experimental Hematology Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy 10Unit of Orthopaedics, IRCCS San Raffaele Scientific Institute, Milan, Italy 11Neuroradiology Department and Neuroradiology Research Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy 12Immunogenetics Laboratory, Department of Immunohematology & Blood Transfusion, IRCCS San Raffaele Scientific Institute, Milan, Italy 13MolMed S.p.A., Milan, Italy 14University San Raffaele Vita e Salute, Milan, Italy 15Unit of Anesthesiology, IRCCS San Raffaele Scientific Institute, Milan, Italy 16Department of Health Sciences, University of Genoa, Genoa, Italy 17Department of Radiology, IRCCS San Raffaele Scientific Institute, Milan, Italy *Corresponding author. Tel: +44 1613062526; E-mail: [email protected] *Corresponding author. Tel: +39 226433036; E-mail: [email protected] *Corresponding author. Tel: +39 255033874; E-mail: [email protected] *Corresponding author. Tel: +39 226432349; E-mail: [email protected] EMBO Mol Med (2015)7:1513-1528https://doi.org/10.15252/emmm.201505636 Correction(s) for this article Intra-arterial transplantation of HLA-matched donor mesoangioblasts in Duchenne muscular dystrophy01 December 2016 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Intra-arterial transplantation of mesoangioblasts proved safe and partially efficacious in preclinical models of muscular dystrophy. We now report the first-in-human, exploratory, non-randomized open-label phase I–IIa clinical trial of intra-arterial HLA-matched donor cell transplantation in 5 Duchenne patients. We administered escalating doses of donor-derived mesoangioblasts in limb arteries under immunosuppressive therapy (tacrolimus). Four consecutive infusions were performed at 2-month intervals, preceded and followed by clinical, laboratory, and muscular MRI analyses. Two months after the last infusion, a muscle biopsy was performed. Safety was the primary endpoint. The study was relatively safe: One patient developed a thalamic stroke with no clinical consequences and whose correlation with mesoangioblast infusion remained unclear. MRI documented the progression of the disease in 4/5 patients. Functional measures were transiently stabilized in 2/3 ambulant patients, but no functional improvements were observed. Low level of donor DNA was detected in muscle biopsies of 4/5 patients and donor-derived dystrophin in 1. Intra-arterial transplantation of donor mesoangioblasts in human proved to be feasible and relatively safe. Future implementation of the protocol, together with a younger age of patients, will be needed to approach efficacy. Synopsis This study reports a safe, first-in-human mesoangioblast cell therapy to treat Duchenne muscular dystrophy (DMD) in 5 young patients, using a successful preclinical strategy, as an exploratory non-randomized open-label phase I–IIa clinical trial of intra-arterial HLA-matched donor cell transplantation. Five patients affected by DMD were treated by intra-arterial infusions of escalating doses of HLA-matched donor mesoangioblasts. The trial was overall safe but showed minimal, if any, efficacy. Data analysis suggested to treat patients at an earlier stage of the disease, optimize in in vitro models each single step of the transplantation protocol, and use genetically corrected, autologous mesoangioblasts in a future trial. Introduction Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy, due to mutations of the X-linked dystrophin gene (Hoffman et al, 1987; Mercuri & Muntoni, 2013a). It causes a progressive degeneration of skeletal and cardiac muscle, leading the patient to reduced motility, wheelchair confinement, and early death, usually due to cardiac and/or respiratory failure (Muntoni et al, 2003; Davies & Nowak, 2006). Drug and physical therapy have extended patients' life span but only modestly its quality (Manzur et al, 2008; Manzur & Muntoni, 2009). A number of new therapies for DMD have entered clinical development, and some have progressed to phase III (Benedetti et al, 2013; Leung & Wagner, 2013; Mercuri & Muntoni, 2013b; Ruegg, 2013; Bushby et al, 2014; Leung et al, 2014; Seto et al, 2014; Voit et al, 2014; Witting et al, 2014; Buyse et al, 2015). All appear to be safe but are often limited to a subset of patients, and long-lasting efficacy has still to be reached. In the past, we have characterized mesoangioblasts (MABs), a subset of pericytes, from mouse, dog, and human skeletal muscle that can be expanded in culture and maintain the ability to differentiate into skeletal and smooth muscle (Minasi et al, 2002; Dellavalle et al, 2007, 2011). Noteworthy, MABs are able to cross the vessel wall when delivered intra-arterially and can thus be distributed to downstream tissues, provided that inflammation and consequent activation of the endothelium are present, as in disease progression of DMD. We tested a protocol of cell therapy in four murine and one canine model of muscular dystrophies, demonstrating safety and partial efficacy of this approach (Sampaolesi et al, 2003, 2006; Diaz-Manera et al, 2010; Tedesco et al, 2011, 2012; Domi et al, 2015). Following an observational study in 28 DMD patients (Pts) aimed at defining longitudinally the disease progression (Lerario et al, 2012), five were enrolled to a "first-in-human" phase I/IIa trial of HLA-matched sibling donor MABs under immunosuppression (Eudract 2011-000176-33). Results Intra-arterial mesoangioblast infusions Five patients (details in Table 1) underwent transplantation of MABs obtained from a muscle biopsy of an HLA-matched brother. Target dose of MABs was consistent with doses administered to dystrophic dogs in preclinical tests (Sampaolesi et al, 2006). All patients received at least four infusions (Appendix Fig S1) in upper and lower (Pt 01, Pt 02, Pt 03) or only lower limbs (Pt 05, Pt 06), under immunosuppression regimen (tacrolimus). The cells used as MP underwent a number of controls before infusion and showed the features reported in Appendix Table S1. In essence, they were still able of good proliferation, expressed the expected phenotype (Tonlorenzi et al, 2007), and had variable ability to differentiate into multinucleated myotubes in culture. Table 1. Patient clinical features Pt 01 Pt 02 Pt 03 Pt 05 Pt 06 Dystrophin mutations Exon deletion 47–52 Exon deletion 4–44 Exon deletion 45–50 Point mutation Exon 43 Point mutation Exon 59 Steroid (mg/kg) Deflazacort 0.75/every other day Deflazacort 0.7/every other day Prednisone 0.5/daily Deflazacort 0.9/every other day Deflazacort 0.5/every other day Age at first infusion (year) 12.4 8.5 9.6 9.2 12.2 Loss of ambulation (age) 12 10.2 10.8 Preserved walk ability 12.1 Weight (kg)a 50 25 37 38 46 Height (cm)a 137 123 140 126 160 Cardiac function Normal Normal Normal Normal Normal Lung function Normal Normal Normal Normal Normal Other comorbidities None None None None None a Measured at first infusion. MAB infusions were in general well tolerated; however, one SAE, a thalamic stroke in Pt 03, was observed out of 23 infusions (see below and Appendix Table S2 for details). In Pt 01 and Pt 03, cell dose was inferior to target. Immediately after MAB infusion, an asymptomatic cutaneous reticulum (livedo reticularis) appeared in the left abdominal lower quadrant (Fig 1A) in Pt 01 and disappeared spontaneously after 1 day; it was attributed to the infusion of small cell clumps, occasionally appearing in confluent cultures (Fig 1B). Also, Pt 02 showed a transient livedo reticularis after two MAB infusions (in left hand and left limb; Fig 1C). More details and a comparison with healthy children of the same age are reported in the legend to Appendix Table S2. To avoid the occurrence of cell clumps, we amended the protocol to allow filtration of the MP with a 70-μm cell strainer. Figure 1. Side effects of MAB treatment of DMD patients Livedo reticularis in the left abdominal lower quadrant after the first infusion in Pt 01. Small clump of MABs observed in the first preparation of MP before the infusion of Pt 01. Scale bar, 30 μm. Livedo reticularis in the left hand of Pt 02 after the first infusion. Brain MRI acquired 1 day after the MAB infusion showing acute small thalamic stroke in Pt 03. Axial diffusion-weighted imaging (left) and fluid-attenuated inversion recovery (FLAIR; right) images show a focal spot of hyperintensity within the right thalamus consistent with acute stroke. FLAIR MRI axial image obtained in Pt 03 1 month after the acute stroke, showing the expected evolution of the right thalamic lesion. Download figure Download PowerPoint In Pt 03, during the first MAB infusion, the pre-infusion diagnostic angiography of the right lower limb revealed contrast inflow delay, likely due to vasospasm of the ipsilateral iliac–femoral arterial axis. The patient was thus infused on the contralateral patent artery after iliac crossing; the vasospasm resolved after injection of vasodilator. Pt 03 showed one SAE after the fourth (last) infusion. Five hours after MAB infusion, the Pt had an episode of vomiting and atrial fibrillation was revealed (but we do not know when it started since the Pt had not been monitored after the infusion), which resolved spontaneously one hour after having being detected. ECG, echocardiography, and color Doppler ultrasound of arteries at four limbs were all normal. The subsequent night, he had headache, photophobia, and vomiting, which solved with paracetamol. Neurological examination was normal, but brain MRI showed an acute thalamic stroke (Fig 1D). Intracranial arterial and venous MR angiography (MRA) and contrast-enhanced MRA of the supra-aortic arteries showed normal caliber and flow signal of the examined vessels. Transcranial Doppler ultrasound with micro-bubbles was normal. He was started on oral aspirin and no further complication occurred. Cerebral MRI 1 month later showed normal evolution of the ischemic lesion (Fig 1E). No new lesions or any clinical consequences were detected. Due to the stroke in Pt 03, study Data Safety Monitoring Board (DSMB) recommended in Pt 05 and Pt 06 MAB infusions only in lower limbs for safety and with the intention to increase cell dose to reach target treatment in lower limbs. No SAEs were observed in these last patients (10 infusions). Donor cell engraftment and dystrophin expression Muscle biopsies performed 2 months after the last infusion showed histological features of muscular dystrophy in all patients (Fig 2A and B). Fiber regeneration (identified by anti-fetal myosin) was minimal, ranging from 3 to 32% (Fig 2C), and rather low as compared to those usually observed in younger DMD patients (50–60%). The DNA chimerism analysis revealed minimal donor cell engraftment, ranging from 0.00 to 0.69% (Appendix Table S3). Figure 2. Muscle biopsies of DMD-treated patients Hematoxylin and eosin staining of muscle biopsies from Pt 01, Pt 02, and Pt 03 performed 2 months after the last MAB infusion. Images show diffuse increase of connective tissue, atrophic and hypertrophic fibers, degenerating fibers, and diffuse centralization of nuclei. Scale bar, 100 μm. Hematoxylin and eosin staining of muscle biopsies from Pt 05 and Pt 06 performed 1 month before the first MAB infusion and 2 months after the last MAB infusion. Images show diffuse increase of connective tissue, atrophic and hypertrophic fibers, degenerating fibers, and diffuse centralization of nuclei. No evident differences were observed between biopsy performed after and before treatment. Scale bar, 100 μm. Immunohistochemistry showing fetal myosin expression in muscle biopsy of DMD patients performed 2 months after the last MAB infusion. The staining was performed in tibialis anterior muscle for Pt 01, Pt 02, and Pt 03 and in gastrocnemius muscle for Pt 05 and Pt 06. A higher number of positive (brownish staining) fibers (representing regenerating fibers) were observed in Pt 02, Pt 05, and Pt 06. Scale bar, 100 μm. Download figure Download PowerPoint Pt 01 and Pt 03 showed virtually no dystrophin expression by immunohistochemistry (Fig 3A). Pt 02 showed scattered, faint, dystrophin positivity in some muscle fibers in post-treatment biopsies. Fiber staining was discontinuous, but revealed also with anti-dys1 antibody, which recognizes a portion of deleted protein absent in revertant fibers (Fig 3B). Pt 05 and Pt 06 showed some fibers positive for dystrophin in both pre- and post-treatment samples (Fig 3C and D). We then applied semi-quantitative measurement of dystrophin expression levels comparing pre-treatment muscle of Pt 01 (sample of muscle obtained from the biopsy performed at time of diagnosis), Pt 05 and Pt 06 (muscle biopsy performed before MAB therapy) with levels in post-treatment muscle. Pt 05 showed modest post-treatment increase of dystrophin levels with anti-dys2 antibody, as mean dystrophin fluorescence intensity increased from 3 to 11% of normal control after treatment. Pt 01 and Pt 06 did not show any increase in protein expression (Appendix Fig S2). However, similar quantification with anti-dys1 antibody did not show any increase in dystrophin levels in Pt 01 and Pt 05, whereas a modest increase was observed in Pt 06 (from 11 to 22%; Appendix Fig S2). Figure 3. Effects of MAB treatment on dystrophin expression Confocal immunofluorescence of muscle biopsy from Pt 01 (tibialis anterior post-treatment) stained with anti-dystrophin dys1 antibody (which recognizes protein fragment encoded by exons 26–30, green signal) and anti-laminin-2 (to delineate muscle fibers, red signal); DAPI identifies nuclei (blue signal). In the left image, only anti-dystrophin staining is shown. No dystrophin-positive fibers were observed. Scale bar, 100 μm. Confocal immunofluorescence of muscle biopsy from Pt 02 (tibialis anterior post-treatment) stained with anti-dystrophin dys1 antibody (green signal) and anti-laminin-2 (red signal); DAPI identifies nuclei (blue signal). In the left image, only anti-dystrophin staining is shown. Some fiber shows mild and discontinuous dystrophin staining. Scale bar, 100 μm. Immunofluorescence of muscle biopsy from Pt 05 taken before (left, gastrocnemius) and after treatment (right, gastrocnemius) stained with anti-dystrophin dys2 antibody (which recognizes exons 77–79). The number and intensity of dystrophin-positive fibers is increased in the post-treatment biopsy. Scale bar, 80 μm. Immunofluorescence of muscle biopsy from Pt 06 taken before (left, gastrocnemius) and after treatment (right, gastrocnemius) stained with anti-dystrophin dys2 antibody. Few fibers show scattered dystrophin staining, without obvious differences between pre- and post-treatment samples. Scale bar, 80 μm. Results of Western blot analysis involving dystrophin antibodies dys1, Mandys18, and Manex46e (recognizing respectively exons 26–30, 17–35, and 46), of total protein extracts (20 μg) obtained from post-treatment biopsy specimens of Pt 01 (tibialis a
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