Poster abstracts
Poster number 15 submitted by Shannon Wright
Blocking RAN translation enhances FMRP expression and reduces toxicity in Fragile X stem cells.
Shannon E. Wright (Neuroscience Graduate Program, University of Michigan, Ann Arbor, MI), Caitlin M. Rodriguez, Jill M. Haenfler, Yu Lui (Department of Neurology, University of Michigan, Ann Arbor, MI), Michael A. Sutton (Molecular & Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI), Frank Rigo (Ionis Pharmaceuticals, San Diego, CA), Jack M. Parent, Sami J. Barmada (Department of Neurology, University of Michigan, Ann Arbor, MI), Peter K. Todd (Department of Neurology, University of Michigan, Ann Arbor, MI)
Abstract:
Expansion of a CGG repeat in the 5’ UTR of the FMR1 gene underlies a heterogeneous set of human clinical disorders, including Fragile X Syndrome and Fragile X-Associated Tremor/Ataxia Syndrome. While full mutations (>200 repeat expansions) result in FXS typically lead to methylation and silencing of FMR1 expression, patients often display mosaicism of FMR1 methylation and CGG repeat length in individual cells. Large transcribed CGG repeats are potentially toxic as RNA or by triggering Repeat associated non-AUG initiated translation (CGG RAN). Moreover, large repeats can impede translation of FMRP even when transcription is sufficient. Therefore, effective therapies for Fragile X-associated disorders need to simultaneously block CGG RAN and enhance production of FMRP. To this end, our group recently developed a series of antisense oligonucleotides that selectively target RAN initiation sites (RAN ASOs) on the FMR1 transcript. In reporter systems and human cell lines, these ASOs suppress RAN translation at both normal and expanded repeat sizes. Surprisingly, this loss of CGG RAN is associated with a marked increase in endogenous FMRP expression, suggesting that CGG RAN acts normally to inhibit FMRP synthesis. Using patient-derived induced pluripotent stem cells (iPSCs) from an unmethylated full mutation (UFM) carrier, we generated human neurons that exhibit normal FMR1 mRNA transcription but very low FMRP expression. Application of RAN ASOs on these neurons effectively reduced accumulation of CGG RAN products and enhanced neuronal FMRP expression. These biochemical correction were associated with enhanced UFM neuronal survival. Together, these data suggest a native function for CGG RAN in regulating FMRP synthesis and demonstrate that targeting CGG RAN has the potential to correct multiple disease relevant features in Fragile X-associated disorders.
Keywords: RAN translation, Fragile X, iPSCs