RESUMEN
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.
Asunto(s)
Distrofina/genética , Mutación del Sistema de Lectura , Edición Génica/métodos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , ARN Guía de Kinetoplastida/genética , Animales , Modelos Animales de Enfermedad , Exones , Femenino , Regulación de la Expresión Génica , Terapia Genética , Genoma , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/terapia , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Espectrometría de Masas , Músculo Esquelético/metabolismo , Músculos/metabolismo , Mioblastos/metabolismo , Miocitos Cardíacos/metabolismo , Proteoma , PorcinosRESUMEN
OBJECTIVES: Bronchoscopy is an integral part of pulmonary medicine. In recent years, a series of new technologies have evolved. It is to assume that significant changes have also occurred in clinical practice. We conducted a nationwide survey to evaluate the current status of care and to compare it with earlier reports. METHODS: A standard questionnaire was sent to 1875 institutions to assess the clinical practice of bronchoscopy in Germany with respect to general issues, education, sedation/anaesthesia and technical aspects. RESULTS: The returned questionnaires cover 301,965 bronchoscopies, performed by 2158 physicians over 12 months, making it the largest survey to date. The proportion of rigid bronchoscopies has decreased and amounts to 7.3% at present. Atropine as a premedication is hardly used any more. Sedation is routinely applied in 88% of flexible bronchoscopies, for which a combination of propofol and midazolam is preferred by most institutions (41.3%), followed by propofol monotherapy (28.3%). 74.4% of institutions accept aspirin for transbronchial biopsy, 8.1% dual platelet inhibition. 62.4% of all institutions perform airway recanalisation, favouring cryotherapy and argon plasma coagulation. 9.1% of bronchoscopies are supported by endobronchial ultrasound. CONCLUSION: Compared to preceding surveys, the experience of bronchoscopists, especially regarding interventional procedures, has increased. Endobronchial ultrasound has become a standard of care, as has patient sedation with propofol.