Poster abstracts

Poster number 117 submitted by Rafia Rahat

AAV-delivered Splice Switching Oligonucleotides: Restoring p53 function through MDM2 splicing modulation

Rafia Rahat (Center for Childhood Cancer Research, Nationwide Childrens Hospital and Department of Pediatrics and the Center for RNA Biology, The Ohio State University), Matias Montes (Department of Chemical and Systems Biology, Stanford University), Akila S Venkataramany (Center for Childhood Cancer Research, Nationwide Childrens Hospital and Department of Pediatrics and the Center for RNA Biology, The Ohio State University), Ravi Dhital (Center for Childhood Cancer Research, Nationwide Childrens Hospital and Department of Pediatrics, The Ohio State University), Kevin Cassady (Center for Childhood Cancer Research, Nationwide Childrens Hospital and Department of Pediatrics and the Center for RNA Biology, The Ohio State University), Dawn Chandler (Center for Childhood Cancer Research, Nationwide Childrens Hospital and Department of Pediatrics and the Center for RNA Biology, The Ohio State University)

Abstract:
MDM2 negatively regulates the tumor suppressor p53, but its alternatively spliced isoform MDM2-ALT1 counteracts this by inhibiting full-length MDM2 (MDM2-FL). MDM2 is upregulated and drives oncogenesis in multiple sarcomas. Switching splicing from MDM2-FL to MDM2-ALT1 can restore p53 function and impede cancer progression. Our lab has shown that Splice Switching Oligonucleotides (SSOs) induce MDM2-ALT1 expression and reactivate p53 function. For in vivo target specific delivery, we expressed our SSO sequence as an antisense-RNA from a lentiviral plasmid and packaged it inside extracellular vesicles (EV). These SSO/antisense-RNA packaged in EVs successfully switched splicing and produced MDM2-ALT1. However, this effect was transient, likely due to degradation of the EV encapsulated antisense-RNA. To enhance stability, we plan to attach the SSO sequence to modified U7 snRNA and expressed it as a transgene from adeno-associated viral vectors (AAVs). We hypothesize that these engineered AAVs will facilitate in vivo delivery of SSOs to p53 wild-type tumor cells, induce MDM2-ALT1 expression, and restore p53 activity to reverse the cancer phenotype. We incorporated our SSO sequence in an AAV viral vector to be expressed as a transgene and validated its ability to induce MDM2-ALT1 splicing in vitro using RT-PCR. Next, we will construct viruses to express this transgene for in vivo delivery in xenograft tumor models. Additionally, we plan to package SSO/antisense-RNA attached to U7 snRNA in EVs to validate potentially increase in stability and target-specific delivery. The transgene expressed by the AAV viral vector successfully induced MDM2-ALT1 expression and stem-loop RT-qPCR analysis confirmed the packaging of the SSO/antisense-RNA within EVs collected from AAV vector transfected RH30 cells. We believe AAV or EV-mediated delivery of SSOs to p53 wild-type tumor cells will induce MDM2-ALT1 expression, enhancing p53 activity and potentially reversing the cancer phenotype.

References:
1. Comiskey, Daniel F Jr et al. “SRSF2 Regulation of MDM2 Reveals Splicing as a Therapeutic Vulnerability of the p53 Pathway.” Molecular cancer research : MCR vol. 18,2 (2020): 194-203. doi:10.1158/1541-7786.MCR-19-0541
2. Uchikawa, Hideki et al. “U7 snRNA-mediated correction of aberrant splicing caused by activation of cryptic splice sites.” Journal of human genetics vol. 52,11 (2007): 891-897. doi:10.1007/s10038-007-0192-8
3. Gadgil, Ankur, and Katarzyna Dorota Raczyńska. “U7 snRNA: A tool for gene therapy.” The journal of gene medicine vol. 23,4 (2021): e3321. doi:10.1002/jgm.3321

Keywords: Alternative splicing, Splice Switching Oligonucleotides, AAV mediated SSO delivery