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Recent progress in CRISPR gene editing tools has substantially increased the opportunities for curing devastating genetic diseases. Here we compare in-frame deletion by CRISPR-based non-homologous blunt end joining (NHBEJ), homology-directed repair (HDR), and prime editing (PE, PE2, and PE3)-based correction of two Duchenne Muscular Dystrophy (DMD) loss-of-function mutations (c.5533G>T and c.7893delC). To enable accurate and rapid evaluation of editing efficiency, we generated a genomically integrated synthetic reporter system (VENUS) carrying the DMD mutations. The VENUS contains a modified enhanced green fluorescence protein ( EGFP ) gene, in which expression was restored upon the CRISPR-mediated correction of DMD loss-of-function mutations. We observed that the highest editing efficiency was achieved by NHBEJ (74-77%), followed by HDR (21-24%) and PE2 (1.5%) in HEK293T VENUS reporter cells. A similar HDR (23%) and PE2 (1.1%) correction efficiency is achieved in fibroblast VENUS cells. With PE3 (PE2 plus nicking gRNA), the c.7893delC correction efficiency was increased 3-fold. Furthermore, an approximately 31% correction efficiency of the endogenous DMD: c.7893delC is achieved in the FACS-enriched HDR-edited VENUS EGFP+ patient fibroblasts. We demonstrated that a highly efficient correction of DMD loss-of-function mutations in patient cells can be achieved by several means of CRISPR gene editing.

Background: The aim of this study was to demonstrate the efficacy of a management model to reduce hospitalizations in patients recently admitted for acute decompensated heart failure., Methods: The management model was based on a close integration between hospital and territory health services. Clinical evaluation, ECG, echocardiographic findings, total body bioimpedance and brain natriuretic peptide serum levels were used to assess clinical stability of patients at discharge and during follow-up. A dedicated nurse (care manager) was involved in patient empowerment, telephone follow-up, check of adherence to therapy and clinical conditions, and management of scheduled evaluations. All hospitalizations occurring prior to or after enrolment in the municipalities involved in the study were considered, as well as the hospitalizations occurring among enrolled patients., Results: A total of 301 patients were enrolled, 226 of them from the Centers of the Puglia Region, Italy. An overall reduction of 6% in total hospitalizations with DRG 127 was observed; the reduction was most evident in the two centers with the smaller reference population (-16% and -26%, respectively). In the group of patients enrolled, an overall reduction in hospital admissions was observed after comparing the period before and after enrolment. A significant increase in the prescription rate of angiotensin-converting enzyme inhibitor/angiotensin receptor blockers, beta-blockers and aldosterone antagonists was also observed. Finally, an overall reduction in the costs for patient management was demonstrated. Similar results were obtained in the two additional Centers of the Basilicata and Lazio Regions., Conclusions: An integrated management between hospital and territory allows optimization of the management of heart failure patients, with improvement of therapy and reduction in hospitalizations and management costs.