Plasmid pcDNA3.1(+)-P2A-eGFP from GenScript Biotech Corporation has been engineered to include SBDS wild-type cDNA sequence followed by the self-cleavage P2A sequence and the eGFP gene as positive control of the transfections. The plasmid has then been mutagenized to introduce the c.183_184TA>CT mutation in the SBDS cDNA sequence. HEK293T cells have been transfected with Polyplus jetOPTIMUS following the manufacturer's indications. Fluorescence microscopy was performed with an Olympus IX51 Inverted fluorescent microscope, equipped with U-RFL-T200 lamp and UTV0.5XC-3 CCD Camera (Olympus). Image analysis was performed by analSYS^B 5.0 (Soft Imaging System)

Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, exocrine pancreatic insufficiency and skeletal abnormalities. SDS bone marrow haematopoietic progenitors show increased apoptosis and impairment in granulocytic differentiation. Loss of Shwachman-Bodian-Diamond syndrome (SBDS) expression results in reduced eukaryotic 80S ribosome maturation. Biallelic mutations in the SBDS gene are found in ~90% of SDS patients, ~55% of whom carry the c.183-184TA>CT nonsense mutation. Several translational readthrough-inducing drugs aimed at suppressing nonsense mutations have been developed. One of these, ataluren, has received approval in Europe for the treatment of Duchenne muscular dystrophy. We previously showed that ataluren can restore full-length SBDS protein synthesis in SDS-derived bone marrow cells. Here, we extend our preclinical study to assess the functional restoration of SBDS capabilities in vitro and ex vivo. Ataluren improved 80S ribosome assembly and total protein synthesis in SDS-derived cells, restored myelopoiesis in myeloid progenitors, improved neutrophil chemotaxis in vitro and reduced neutrophil dysplastic markers ex vivo. Ataluren also restored full-length SBDS synthesis in primary osteoblasts, suggesting that its beneficial role may go beyond the myeloid compartment. Altogether, our results strengthened the rationale for a Phase I/II clinical trial of ataluren in SDS patients who harbour the nonsense mutation.