Poster abstracts
Poster number 44 submitted by Rafia Rahat
MDM2 pre-mRNA: A target for RNA therapy
Rafia Rahat (Center for Childhood Cancer Research, Nationwide Childrens Hospital and Molecular, Cellular and Developmental Biology Program, The Ohio State University), Matias Montes (Department of Chemical and Systems Biology, Stanford University), Uksha Saini (Center for Childhood Cancer Research, Nationwide Childrens Hospital), Kevin Cassady (Center for Childhood Cancer Research, Nationwide Childrens Hospital and Department of Pediatrics, The Ohio State University), Dawn S 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 negative regulation of p53 by inhibiting full-length MDM2 (MDM2-FL). MDM2 is upregulated and a driver of oncogenesis in multiple sarcomas and is therefore an attractive therapeutic target. Switching splicing from MDM2-FL to MDM2-ALT1 can restore p53 function and impede cancer progression. Previous research from our lab has shown that Splice Switching Oligonucleotides (SSOs) induce MDM2-ALT1 expression and reactivate p53 function. However, achieving in vivo delivery of these SSOs to tumors has been challenging. To address this, we aim to develop SSOs with increased stability and prolonged activity by incorporating the SSO sequence into adeno-associated viral vectors (AAVs) to express them as transgene. We hypothesize that the engineered AAVs will facilitate in vivo delivery of these SSOs to p53 wild-type tumor cells, will induce MDM2-ALT1 expression, and restore p53 activity to reverse the cancer phenotype.
We have shown that extracellular vesicles (EV) can deliver cargo in specific target tissue, so we packaged our SSO in EVs for in vivo delivery. EV-packaged SSOs initially modulated MDM2 splicing but required repeated administrations due to short-lived activity. To bolster stability, we incorporated a stability sequence to sequester the SSOs inside nucleus providing protection from cytoplasmic exonucleases. However, this approach failed to extend their lifespan. Therefore, we plan to incorporate our SSO as transgene in AAV viral vector and validate the ability of virally expressed SSOs in inducing MDM2-ALT1 splicing in vitro. We will also package this SSO expressed as transgene in AAVs and test their efficacy in xenograft tumors in vivo. To enable multiple administrations without inducing immunogenicity from the AAV-mediated delivery, we eventually plan to package the AAVs into EVs to evade the immune system and achieve target specific delivery.
References:
1. Comiskey, Daniel F Jr et al., Molecular cancer research: MCR, 2020
2. Chandler, Dawn S et al., Cancer research, 2006
3. Do Minh, Aline et al., Viruses, 2021
4. O’Brien, Killian, et al., Nature reviews Molecular cell biology, 2020
5. Bisset, Darren R et al., Human molecular genetics, 2015
Keywords: Alternative splicing, Splice Switching Oligonucleotides, AAV mediated therapeutic delivery