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

Abstract

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.

Lethal Complications and Complex Genotypes in Shwachman Diamond Syndrome: Report of a Family with Recurrent Neonatal Deaths and a Case-Based Brief Review of the Literature

“…However, studies have shown that, in ~10% of the transcribed RNA, normal splicing occurs and retains some of the functional SBDS protein, which may explain the high survival rates of c.258+2T>C homozygous embryos [62]. More recent findings, showing that exon 2 is rich in splicing regulatory elements and cryptic splice sites, confirmed aberrant splicing mechanisms, whereby transcripts lacking exon 2 were traced in control cells from normal subjects, and correct transcripts (~2%), allowing residual SBDS expression, were traced in cells with the variant [64]. SBDS:c.242C>G, a novel variant of unknown significance, is located in exon 2 and causes a threonine-to-serine substitution at residue 81 p.(Thr81Ser) located within the N-terminal domain (FYSH) of the protein.…”

“…Ataluren (PTC124) and an additional NV848 analogue were shown to restore full-length SBDS protein synthesis [ 98 ]. In addition, counteracting the other common c.258+2T>C variant by RNA therapeutics and Base/Prime Editing was currently proposed as the basis of an “SDS personalized therapy” using edited hematopoietic stem cells [ 64 ].…”

Development of Engineered-U1 snRNA Therapies: Current Status

“…All modified U1 snRNAs strongly promoted the use of a proximal cryptic exonic 5 ss and two ExSpeU1s also promoted the use of an exonic cryptic 3 ss. However, the modified U1 led also to a slight increase in the relative proportion of the corrected transcripts meaning that forcing the definition of the defective exon by using a compensatory U1 snRNA, albeit with low efficacy, did promote the synthesis of correct transcripts [46].…”

“…However, there are also works reporting the correction of mutations affecting the more conserved positions of the 5 ss that is, those located at +1 or +2 sites. To the best of our knowledge, though, there are only three reports on the successful correction of this type of mutation: one for the Fanconi anemia C (FANCC) [45], other for the rare Lysosomal Storage Disease Mucopolysaccharidosis type IIIC (MPS IIIC) [11], and the third one for Shwachman-Diamond Syndrome [46].…”

“…Another example in which positive results were observed came from a recent study [46] involving a very common 5 ss variant that is present in more than 90% [68,69] of worldwide patients with Shwachman-Diamond syndrome, one of the most common inherited bone marrow failure syndromes. The c.258+2T>C variant at the 5 ss of SBDS gene exon 2 is associated with aberrant pre-mRNA splicing leading to exon 2 skipping but trace levels of correct transcripts (2%) are also detected by qPCR, which account for residual protein expression and explain the survival of affected patients [46].…”