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 gene mutations. Only supportive treatments are available, with hematopoietic cell transplantation required when marrow failure occurs. Among all causative mutations, the 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 splicing and showed that 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.