D - Oligonucleotide Therapeutics (including siRNAs, aptamers, antagomirs, miRNAs, shRNA, antisense, and splice switching oligos, plasmids)
335: miRNA Site-Blocking Oligonucleotides as a Novel Therapeutic Strategy for STXBP1 Encephalopathy
Type: Oral Abstract Session
Presentation Details
Session Title: Oligonucleotide Therapeutics
STXBP1 encephalopathy is a rare neurodevelopmental disorder caused by heterozygous mutations in the STXBP1 gene. STXBP1 plays an essential role in neurotransmitter release by regulating pre-synaptic vesicle exocytosis. Loss of function mutations in STXBP1 impair neurotransmission, resulting in epilepsy, intellectual disability and motor and behavioral impairments. In this case, the upregulation of the remaining functional STXBP1 allele may be therapeutic. Since most human mRNAs are at least partly repressed by miRNAs, disrupting the interaction between miRNAs and STXBP1 mRNA could upregulate STXBP1 expression. Here, we performed comprehensive in silico analyses and in cellulo miRNA sequencing to identify multiple miRNAs that are both predicted to bind to STXBP1-3’UTR and highly expressed in STXBP1 patient iPSC-neurons. Further, our CLIP-seq data from human brain tissue showed prominent binding of AGO2 to the same region of the STXBP1-3’UTR in which multiple miRNA candidates are predicted to bind, suggesting miRNA-mediated regulation of STXBP1 mRNA in human brain. In STXBP1 patient iPSC-neurons, blocking miRNA candidates using antagomirs led to STXBP1 mRNA upregulation, thus identifying miRNAs that are repressing STXBP1 in the disease context. As a therapeutic approach, we designed Steric Blocking Oligonucleotides (SBOs) targeting candidate miRNA binding sites in STXBP1-3’UTR and screened them in NGN2-inducible neurons, identifying SBOs that increased STXBP1 mRNA abundance. Also, we showed that several “lead” SBOs were capable of augmenting STXBP1 mRNA abundance in a dose-dependent manner in STXBP1 patient iPSC-neurons, and demonstrated that these compounds act by specifically disrupting the binding of the candidate miRNAs to STXBP1-3’UTR. Finally, we have engineered and characterized the first STXBP1 humanized mouse model in which we replaced the mouse Stxbp1 locus with the entire human STXBP1 gene. We are currently generating a STXBP1 humanized haploinsufficient mouse model, which represents an ideal platform for therapeutic testing and functional rescue assessment in vivo. Overall, these studies reveal several miRNA site-blocking oligonucleotides as promising therapeutic candidates for the treatment of STXBP1-related disorders.
Alex J. Felix1,2, Jennine M. Dawicki-McKenna1,2, Elisa A. Waxman2,3, Congsheng Cheng3, Alexey Bogush1,2, Lea V. Dungan2,3, Deborah L. French2,3, Beverly L. Davidson2,3, Benjamin L. Prosser1,2
1University of Pennsylvania, Philadelphia, PA,2Center for Epilepsy and NeuroDevelopmental Disorders (ENDD), Philadelphia, PA,3Children’s Hospital of Philadelphia, Philadelphia, PA"
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