Volumetric muscle loss (VML) is associated with loss of skeletal muscle function, and current treatments show limited efficacy. Here we show that bioconstructs suffused with genetically-labelled muscle stem cells (MuSCs) and other muscle resident cells (MRCs) are effective to treat VML injuries in mice. Imaging of bioconstructs implanted in damaged muscles indicates MuSCs survival and growth, and ex vivo analyses show force restoration of treated muscles. Histological analysis highlights myofibre formation, neovascularisation, but insufficient innervation. Both innervation and in vivo force production are enhanced when implantation of bioconstructs is followed by an exercise regimen. Significant improvements are also observed when bioconstructs are used to treat chronic VML injury models. Finally, we demonstrate that bioconstructs made with human MuSCs and MRCs can generate functional muscle tissue in our VML model. These data suggest that stem cell-based therapies aimed to engineer tissue in vivo may be effective to treat acute and chronic VML.

Type I mucopolysaccharidosis (MPS I) IntroductionType I mucopolysaccharidosis (MPS I) is one of the most frequent lysosomal storage disorders (LSDs) and is due to the inherited deficiency of ␣-L-iduronidase (IDUA) activity, which results in the accumulation of its unprocessed substrates (glycosaminoglycans; GAGs) in many organs. 1 The disorder is systemic and clinically heterogeneous. Clinical manifestations include skeletal dysplasia, joint stiffness, visual and auditory defects, cardiac insufficiency, hepatosplenomegaly, and mental retardation. The clinical spectrum ranges from the severe Hurler syndrome (MPS I-H) to the attenuated Scheie syndrome. Mental retardation is distinctive only of MPS I-H, which is fatal in childhood if untreated, thus representing the variant with the most urgent need for new therapies. Enzyme replacement therapy (ie, parenteral administration of exogenous enzyme that can be internalized by tissue cells via the mannosium-6-phosphate receptor) is recommended only for MPS I patients without primary neurologic disease, due to the inability of the enzyme to efficiently cross the blood-brain barrier; moreover, neutralizing antibodies can attenuate its efficacy. 2 When performed at early ages, hematopoietic stem cell (HSC) transplantation (HCT) from healthy donors alleviates most disease manifestations in MPS I-H patients, likely by migration of the transplant-derived leukocytes into organs, where they can clear the storage and secrete the functional enzyme for correction of the metabolic defect in resident cells. 3 However, despite recent improvements in the outcome of HCT, the morbidity and mortality associated with the procedure are still not negligible, mostly due to rejection and graft-versus-host disease. Moreover, the amount of enzyme that transplantation can provide to the organism can be limiting, especially since donors are often heterozygous siblings. Indeed, a relationship between circulating enzyme levels after transplant and urinary GAGs has been shown 4 : the low enzyme levels achieved with heterozygote donor transplant lead to less adequate reduction in GAG levels. Likely due to partial metabolic correction at disease sites, the impact of HCT on central nervous system (CNS) and skeletal disease, despite being substantial in ameliorating patients' phenotype, could still benefit from further improvement. 5 The benefits of different gene therapy approaches were established in MPS I animal models. Intravenous delivery of viral vectors, which can establish a tissue source for systemic enzyme distribution, was effective in controlling disease manifestations in The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 10, 2018. by guest www.bloodjournal.org From MPS I animal models upon neonatal treatment. [6][7][8][9] However, residu...

IntroductionGlycogenosis type II (Pompe disease, acid maltase deficiency; Online Mendelian Inheritance in Man no. 232300) is an autosomal recessive lysosomal storage disorder caused by acid ␣-glucosidase (GAA) deficiency. The disease is characterized by glycogen storage in liver, spleen, kidney, brain, and endothelial cells and most prominently in skeletal, heart, and smooth muscles. Symptoms arise from muscular weakness and wasting. Infants with complete enzyme deficiency present shortly after birth, lose all muscle strength within 8 months, and succumb to hypertrophic cardiomyopathy and respiratory failure in the first year of life. 1 Children and adults with residual GAA activity show a more protracted course and may become wheelchair bound, dependent on artificial ventilation, and have a shortened life expectancy. Presently, enzyme replacement therapy (ERT) based on intravenous infusion of recombinant human ␣-glucosidase, taken up by mannose 6-phosphate receptor mediated endocytosis, 2,3 is a major therapeutic advance that prolongs the life of affected infants but does not guarantee long-term symptom-free survival, requires biweekly administration, and may induce immune responses to the recombinant protein.As an alternative to ERT, in vivo gene therapy mediated by adenoviral vectors and adeno-associated virus vectors (AAVs) has been investigated in a mouse model of Pompe disease. [4][5][6] However, long-term efficacy can be significantly hampered by antibody formation, 7,8 and adverse immune responses to the vector has been observed after adenoviral and AAV gene therapy in patients. 9,10 For treatment of patients with other lysosomal enzyme deficiencies, allogeneic hematopoietic stem cell (HSC) transplantation has been proposed. 11 HSC transplantation proved effective in ameliorating the neurologic symptoms in murine globoid cell leukodystrophy and human patients 12,13 as well as in mucopolysaccharidosis I (Hurler syndrome). 14,15 Other lysosomal storage disorders such as metachromatic leukodystrophy (MLD) may require higher enzyme levels than provided by HSC transplantation; lentiviral (LV) vector-mediated overexpression of aryl-sulfatase A in HSCs effectively reversed the neuropathologic phenotype in the mouse model. 16 In addition, LV-mediated clinical gene therapy in trial phase of X-linked adrenoleukodystrophy halted progressive cerebral demyelination in 2 patients. 17 Recently, HSC transplantation was shown to promote immune tolerance to ERT in the Pompe mouse model. 18 The use of gene-modified autologous HSCs also overcomes the profound conditioning and immune barriers associated with allogeneic transplantation.The few attempts of HSC transplantation for Pompe disease have not met with success. 19 GAA levels, if any, are low in hematopoietic cells in mice, 18 and allogeneic transplantation is not an obvious treatment. Therefore, high-level vector-driven ectopic expression of the enzyme in hematopoietic cells would be required to accomplish efficacy. We tested the hypothesis that ex vivo LV For persona...

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Pompe disease is an autosomal recessive lysosomal storage disorder characterized by progressive muscle weakness. The disease is caused by mutations in the acid a-glucosidase (GAA) gene. Despite the currently available enzyme replacement therapy (ERT), roughly half of the infants with Pompe disease die before the age of 3 years. Limitations of ERT are immune responses to the recombinant enzyme, incomplete correction of the disease phenotype, lifelong administration, and inability of the enzyme to cross the blood-brain barrier. We previously reported normalization of glycogen in heart tissue and partial correction of the skeletal muscle phenotype by ex vivo hematopoietic stem cell gene therapy. In the present study, using a codon-optimized GAA (GAAco), the enzyme levels resulted in close to normalization of glycogen in heart, muscles, and brain, and in complete normalization of motor function. A large proportion of microglia in the brain was shown to be GAA positive. All astrocytes contained the enzyme, which is in line with mannose-6-phosphate receptor expression and the key role in glycogen storage and glucose metabolism. The lentiviral vector insertion site analysis confirmed no preference for integration near proto-oncogenes. This correction of murine Pompe disease warrants further development toward a cure of the human condition.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by thymidine phosphorylase (TP) deficiency resulting in systemic accumulation of thymidine (d-Thd) and deoxyuridine (d-Urd) and characterized by early-onset neurological and gastrointestinal symptoms. Long-term effective and safe treatment is not available. Allogeneic bone marrow transplantation may improve clinical manifestations but carries disease and transplant-related risks. In this study, lentiviral vector-based hematopoietic stem cell gene therapy (HSCGT) was performed in Tymp−/−Upp1−/− mice with the human phosphoglycerate kinase (PGK) promoter driving TYMP. Supranormal blood TP activity reduced intestinal nucleoside levels significantly at low vector copy number (median, 1.3; range, 0.2–3.6). Furthermore, we covered two major issues not addressed before. First, we demonstrate aberrant morphology of brain astrocytes in areas of spongy degeneration, which was reversed by HSCGT. Second, long-term follow-up and vector integration site analysis were performed to assess safety of the therapeutic LV vectors in depth. This report confirms and supplements previous work on the efficacy of HSCGT in reducing the toxic metabolites in Tymp−/−Upp1−/− mice, using a clinically applicable gene transfer vector and a highly efficient gene transfer method, and importantly demonstrates phenotypic correction with a favorable risk profile, warranting further development toward clinical implementation.

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