Counteracting the Common Shwachman-Diamond Syndrome-Causing SBDS c.258+2T>C Mutation by RNA Therapeutics and Base/Prime Editing.

Shwachman-Diamond syndrome (SDS) represents one of the most common inherited bone marrow failure syndromes and is mainly caused by SBDS gene mutations. Only supportive treatments are available, with hematopoietic cell transplantation required when marrow failure occurs. Among all causative mutations, the SBDS c.258+2T>C variant at the 5' splice site (ss) of exon 2 is one of the most frequent. Here, we investigated the molecular mechanisms underlying aberrant SBDS splicing and showed that SBDS exon 2 is dense in splicing regulatory elements and cryptic splice sites, complicating proper 5'ss selection. Studies ex vivo and in vitro demonstrated that the mutation alters splicing, but it is also compatible with tiny amounts of correct transcripts, which would explain the survival of SDS patients. Moreover, for the first time for SDS, we explored a panel of correction approaches at the RNA and DNA levels and provided experimental evidence that the mutation effect can be partially counteracted by engineered U1snRNA, trans-splicing, and base/prime editors, ultimately leading to correctly spliced transcripts (from barely detectable to 2.5-5.5%). Among them, we propose DNA editors that, by stably reverting the mutation and potentially conferring positive selection to bone-marrow cells, could lead to the development of an innovative SDS therapy.

OTC intron 4 variations mediate pathogenic splicing patterns caused by the c.386G>A mutation in humans and spfash mice, and govern susceptibility to RNA-based therapies.

BACKGROUND: Aberrant splicing is a common outcome in the presence of exonic or intronic variants that might hamper the intricate network of interactions defining an exon in a specific gene context. Therefore, the evaluation of the functional, and potentially pathological, role of nucleotide changes remains one of the major challenges in the modern genomic era. This aspect has also to be taken into account during the pre-clinical evaluation of innovative therapeutic approaches in animal models of human diseases. This is of particular relevance when developing therapeutics acting on splicing, an intriguing and expanding research area for several disorders. Here, we addressed species-specific splicing mechanisms triggered by the OTC c.386G>A mutation, relatively frequent in humans, leading to Ornithine TransCarbamylase Deficiency (OTCD) in patients and spfash mice, and its differential susceptibility to RNA therapeutics based on engineered U1snRNA. METHODS: Creation and co-expression of engineered U1snRNAs with human and mouse minigenes, either wild-type or harbouring different nucleotide changes, in human (HepG2) and mouse (Hepa1-6) hepatoma cells followed by analysis of splicing pattern. RNA pulldown studies to evaluate binding of specific splicing factors. RESULTS: Comparative nucleotide analysis suggested a role for the intronic +10-11 nucleotides, and pull-down assays showed that they confer preferential binding to the TIA1 splicing factor in the mouse context, where TIA1 overexpression further increases correct splicing. Consistently, the splicing profile of the human minigene with mouse +10-11 nucleotides overlapped that of mouse minigene, and restored responsiveness to TIA1 overexpression and to compensatory U1snRNA. Swapping the human +10-11 nucleotides into the mouse context had opposite effects. Moreover, the interplay between the authentic and the adjacent cryptic 5'ss in the human OTC dictates pathogenic mechanisms of several OTCD-causing 5'ss mutations, and only the c.386+5G>A change, abrogating the cryptic 5'ss, was rescuable by engineered U1snRNA. CONCLUSIONS: Subtle intronic variations explain species-specific OTC splicing patterns driven by the c.386G>A mutation, and the responsiveness to engineered U1snRNAs, which suggests careful elucidation of molecular mechanisms before proposing translation of tailored therapeutics from animal models to humans.

Mesenchymal stem cells as adjuvant therapy for limb lengthening in achondroplasia.

Staged leg lengthening allows achondroplastic dwarfs to reach nearly normal height, but it takes long periods of external fixation and it can be burdened by delayed unions. Between 2009 and 2013, eight achondroplastic dwarfs showed delayed unions in the callus formation during femoral lengthening stages in our institute. We performed in-situ injections of bone marrow-derived stem cell concentrates. Patients underwent monthly clinical and radiographic assessment for determination of the healing rate. All eight patients showed an improvement in the regenerated bone, with an average healing index of 23.1 days/cm (range: 18.7-23.8 days/cm). The complete recovery of the delayed consolidation took on an average of 5.2 months (range: 2-10 months). The use of cellular therapy in these patients could represent an innovative application.