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1.
Nat Med ; 26(2): 207-214, 2020 02.
Article in English | MEDLINE | ID: mdl-31988462

ABSTRACT

Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs1, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref. 2) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca2+ handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.


Subject(s)
Dystrophin/genetics , Frameshift Mutation , Gene Editing/methods , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/therapy , RNA, Guide, Kinetoplastida/genetics , Animals , Disease Models, Animal , Exons , Female , Gene Expression Regulation , Genetic Therapy , Genome , Heart Failure/genetics , Heart Failure/therapy , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Male , Mass Spectrometry , Muscle, Skeletal/metabolism , Muscles/metabolism , Myoblasts/metabolism , Myocytes, Cardiac/metabolism , Proteome , Swine
2.
Growth Horm IGF Res ; 51: 6-16, 2020 04.
Article in English | MEDLINE | ID: mdl-31926372

ABSTRACT

OBJECTIVE: Human patients with Duchenne muscular dystrophy (DMD) commonly exhibit a short stature, but the pathogenesis of this growth retardation is not completely understood. Due to the suspected involvement of the growth hormone/insulin-like growth factor 1 (GH/IGF1) system, controversial therapeutic approaches have been developed, including both GH- administration, as well as GH-inhibition. In the present study, we examined relevant histomorphological and ultrastructural features of adenohypophyseal GH-producing somatotroph cells in a porcine DMD model. METHODS: The numbers and volumes of immunohistochemically labelled somatotroph cells were determined in consecutive semi-thin sections of plastic resin embedded adenohypophyseal tissue samples using unbiased state-of-the-art quantitative stereological analysis methods. RESULTS: DMD pigs displayed a significant growth retardation, accounting for a 55% reduction of body weight, accompanied by a significant 50% reduction of the number of somatotroph cells, as compared to controls. However, the mean volumes of somatotroph cells and the volume of GH-granules per cell were not altered. Western blot analyses of the adenohypophyseal protein samples showed no differences in the relative adenohypophyseal GH-abundance between DMD pigs and controls. CONCLUSION: The findings of this study do not provide evidence for involvement of somatotroph cells in the pathogenesis of growth retardation of DMD pigs. These results are in contrast with previous findings in other dystrophin-deficient animal models, such as the golden retriever model of Duchenne muscular dystrophy, where increased mean somatotroph cell volumes and elevated volumes of intracellular GH-granules were reported and associated with DMD-related growth retardation. Possible reasons for the differences of somatotroph morphology observed in different DMD models are discussed.


Subject(s)
Growth Disorders/pathology , Growth Hormone/metabolism , Muscular Dystrophy, Duchenne/pathology , Secretory Vesicles/pathology , Somatotrophs/pathology , Animals , Animals, Genetically Modified , Cell Count , Disease Models, Animal , Dystrophin/genetics , Growth Disorders/complications , Growth Disorders/metabolism , Microscopy, Electron , Muscular Dystrophy, Duchenne/complications , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/metabolism , Organ Size , Pituitary Gland/pathology , Pituitary Gland/ultrastructure , Pituitary Gland, Anterior/pathology , Pituitary Gland, Anterior/ultrastructure , Secretory Vesicles/ultrastructure , Somatotrophs/ultrastructure , Swine
3.
Sci Rep ; 7(1): 3572, 2017 06 15.
Article in English | MEDLINE | ID: mdl-28620237

ABSTRACT

Genetically engineered pigs are a promising source for islet cell transplantation in type 1 diabetes, but the strong human anti-pig immune response prevents its successful clinical application. Here we studied the efficacy of neonatal porcine islet-like cell clusters (NPICCs) overexpressing LEA29Y, a high-affinity variant of the T cell co-stimulation inhibitor CTLA-4Ig, to engraft and restore normoglycemia after transplantation into streptozotocin-diabetic NOD-SCID IL2rγ-/- (NSG) mice stably reconstituted with a human immune system. Transplantation of INSLEA29Y expressing NPICCs resulted in development of normal glucose tolerance (70.4%) and long-term maintenance of normoglycemia without administration of immunosuppressive drugs. All animals transplanted with wild-type NPICCs remained diabetic. Immunohistological examinations revealed a strong peri- and intragraft infiltration of wild-type NPICCs with human CD45+ immune cells consisting of predominantly CD4+ and CD8+ lymphocytes and some CD68+ macrophages and FoxP3+ regulatory T cells. Significantly less infiltrating lymphocytes and only few macrophages were observed in animals transplanted with INSLEA29Y transgenic NPICCs. This is the first study providing evidence that beta cell-specific LEA29Y expression is effective for NPICC engraftment in the presence of a humanized immune system and it has a long-lasting protective effect on inhibition of human anti-pig xenoimmunity. Our findings may have important implications for the development of a low-toxic protocol for porcine islet transplantation in patients with type 1 diabetes.


Subject(s)
Abatacept/genetics , Gene Expression , Immunosuppression Therapy , Islets of Langerhans/metabolism , Animals , Biomarkers , Cell Survival , Gene Knockout Techniques , Heterografts , Humans , Immunity/genetics , Immunohistochemistry , Immunophenotyping , Immunosuppression Therapy/methods , Mice , Mice, Knockout , Mice, Transgenic , Swine
4.
Horm Metab Res ; 47(1): 31-5, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25506683

ABSTRACT

Solid organ and cell transplantation, including pancreatic islets constitute the treatment of choice for chronic terminal diseases. However, the clinical use of allogeneic transplantation is limited by the growing shortage of human organs. This has prompted us to initiate a unique multi-center and multi-team effort to promote translational research in xenotransplantation to bring xenotransplantation to the clinical setting. Supported by the German Research Foundation, an interdisciplinary group of surgeons, internal medicine doctors, diabetologists, material sciences experts, immunologists, cell biologists, virologists, veterinarians, and geneticists have established a collaborative research center (CRC) focusing on the biology of xenogeneic cell, tissue, and organ transplantation. A major strength of this consortium is the inclusion of members of the regulatory bodies, including the Paul-Ehrlich Institute (PEI), infection specialists from the Robert Koch Institute and PEI, veterinarians from the German Primate Center, and representatives of influential ethical and religious institutions. A major goal of this consortium is to promote islet xenotransplantation, based on the extensive expertise and experience of the existing clinical islet transplantation program. Besides comprehensive approaches to understand and prevent inflammation-mediated islet xenotransplant dysfunction [immediate blood-mediated inflammatory reaction (IBMIR)], we also take advantage of the availability of and experience with islet macroencapsulation, with the goal to improve graft survival and function. This consortium harbors a unique group of scientists with complementary expertise under a cohesive program aiming at developing new therapeutic approaches for islet replacement and solid organ xenotransplantation.


Subject(s)
Diabetes Mellitus, Type 1/therapy , Islets of Langerhans Transplantation , Islets of Langerhans/cytology , Transplantation, Heterologous , Animals , Cells, Immobilized/metabolism , Humans , Immune Tolerance/immunology , Islets of Langerhans Transplantation/immunology , Sus scrofa
6.
J Mol Med (Berl) ; 90(5): 597-608, 2012 May.
Article in English | MEDLINE | ID: mdl-22170306

ABSTRACT

Cystic fibrosis (CF) is the most common lethal inherited disease in Caucasians and is caused by mutations in the CFTR gene. The disease is incurable and medical treatment is limited to the amelioration of symptoms or secondary complications. A comprehensive understanding of the disease mechanisms and the development of novel treatment options require appropriate animal models. Existing CF mouse models fail to reflect important aspects of human CF. We thus generated a CF pig model by inactivating the CFTR gene in primary porcine cells by sequential targeting using modified bacterial artificial chromosome vectors. These cells were then used to generate homozygous CFTR mutant piglets by somatic cell nuclear transfer. The homozygous CFTR mutants lack CFTR protein expression and display severe malformations in the intestine, respiratory tract, pancreas, liver, gallbladder, and male reproductive tract. These phenotypic abnormalities closely resemble both the human CF pathology as well as alterations observed in a recently published CF pig model which was generated by a different gene targeting strategy. Our new CF pig model underlines the value of the CFTR-deficient pig for gaining new insight into the disease mechanisms of CF and for the development and evaluation of new therapeutic strategies. This model will furthermore increase the availability of CF pigs to the scientific community.


Subject(s)
Chromosomes, Artificial, Bacterial/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/genetics , Cystic Fibrosis/pathology , Disease Models, Animal , Gene Targeting , Genetic Vectors/genetics , Alleles , Animals , Cells, Cultured , Cystic Fibrosis Transmembrane Conductance Regulator/deficiency , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Fetus/metabolism , Gene Knockout Techniques , Humans , Kidney/metabolism , Kidney/pathology , Male , Mice , Organ Specificity , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sus scrofa
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