Targeting a Putative Intronic Splicing Silencer Salvages Expression from the Recurrent SBDS C.258+2T>C Mutant Allele in Shwachman-Diamond Syndrome Patient Cells and Mouse Model

Shwachman-Diamond syndrome (SDS) is an autosomal recessive inherited bone marrow failure syndrome and leukemia predisposition disorder due to biallelic mutations of SBDS, whose gene product positively regulates 80S ribosome assembly. In addition to predisposition to pancytopenia, myelodysplasia and acute myeloid leukemia, patients frequently experience exocrine pancreatic insufficiency, skeletal dysplasia, cardiac abnormalities, and immune dysfunction. Faithful animal models are lacking to study hematopoiesis and clonal evolution in SDS. Almost all patients carry at least one copy of the SBDS c.258+2T>C mutation that disrupts the intron 2 5' splice site (SS). We hypothesize that this recurrent mutant allele allows for sufficient residual SBDS expression to promote SBDS cellular function and permit organismal development, and thus residual expression could be potentiated through novel therapeutic approaches. Here we present RNA-seq analysis that shows residual expression of full-length SBDS originating from the non-canonical SS of the SBDS c.258+2T>C allele. Most transcripts were the product of aberrant splicing from a cryptic exon 2 SS and exon 2 skipping, both of which result in nonsense transcripts. Prior animal models of SDS have relied on complete or tissue-specific Sbds knock-out and thus fail to replicate the splicing defect and residual SBDS expression. To develop a mouse model to test novel sequence-specific therapeutic strategies, we generated, by CRISPR/Cas9-mediated HDR, mice with a humanized intron 2 5' SS containing the c.258+2T>C mutation at the endogenous mouse Sbds gene (c.199+2T>C). Homozygous Sbds SS mutant knock-in mouse embryos were found to be viable until at least mid-gestation (E14.5) though live births were not observed. In contrast, constitutive Sbds knock-out mice are reported to succumb to early embryonic lethality by E8.51. Using isolated MEFs, we demonstrate that the humanized mouse Sbds mutant allele recapitulates low-level residual expression, aberrant splicing from a cryptic exon 2 SS, and exon 2 skipping, although exon skipping appears to be greater and residual expression lower as compared to the human mutant allele. Consistent with the characteristic SDS molecular defect, homozygous Sbds c.199+2T>C MEFs show impaired 80S ribosome assembly, accumulation of EIF6 on the 60S ribosomal subunit, and induction of a p53-dependent stress response. To explore strategies to further increase residual wild type SBDS transcript expression from the SBDS c.258+2T>C allele, we designed anti-sense oligonucleotides (ASOs) targeting potential splice regulatory sequences. We evaluated 38 antisense oligos, identifying several that block a putative intronic splicing silencer (ISS) that suppresses normal splicing from the non-canonical c.258+2T>C SS, with the most potent ASO resulting in 4.0-fold increase in SBDS expression (p<0.01) in fibroblasts from 4 different SDS patients. Disruption of sequences within this putative ISS by Cas12a nuclease-mediated indels result in 1.4 and 2.4 fold increased SBDS/Sbds expression from SDS patient fibroblasts and homozygous MEFs respectively (p<0.05). Together these studies present a novel mouse model for SDS recapitulating the recurrent SS mutation with residual intact splicing and indicate that salvaging residual SBDS expression by modulating aberrant splicing is a promising strategy for SDS therapeutics. References 1. Zhang, S., Shi, M., Hui, C.C., Rommens, J.M. 2006. Loss of the mouse ortholog of the shwachman-diamond syndrome gene (sbds) results in early embryonic lethality. Cell. Biol.26:6656-6663. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal