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1259 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

Program: Oral and Poster Abstracts
Session: 509. Bone Marrow Failure and Cancer Predisposition Syndromes: Congenital: Poster I
Hematology Disease Topics & Pathways:
Research, Bone Marrow Failure Syndromes, Translational Research, Inherited Marrow Failure Syndromes, Diseases, Therapies
Saturday, December 10, 2022, 5:30 PM-7:30 PM

Michael J. Peters1*, Anna Deck1*, April Hu, MD, PhD2*, Yuko Fujiwara, PhD1*, Jinkuk Kim, PhD2*, Aubrie Soucy, MA2*, Kevin Zhang1*, Julianna Kenny1*, Linda Y. Lin1*, Alyssa L. Kennedy, MD, PhD1, Caitlin Bowers1*, Chad E. Harris, MS1*, Chi Yuan Zhang, PhD1*, Karthik Ponnienselvan3*, Pengpeng Liu, PhD3*, Kevin Luk, PhD3*, Davide Seruggia, PhD4,5*, R. Coleman Lindsley6, John P. Manis, MD1*, Christian Brendel, PhD1*, Scot A. Wolfe, PhD3*, Stuart H. Orkin, MD1, Timothy Yu, MD, PhD2*, Akiko Shimamura1 and Daniel E. Bauer, MD, PhD1

1Division of Hematology/Oncology, Boston Children's Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA
2Division of Genetics and Genomics, Boston Children’s Hospital, Harvard Medical School, Boston, MA
3Department of Molecular, Cell and Cancer Biology, Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA
4CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
5St. Anna Children’s Cancer Research Institute (CCRI), Vienna, Austria
6Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA

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.


  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.

Disclosures: No relevant conflicts of interest to declare.

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