Mentor Name: Steven Jonas

Our ALSF POST project focuses on developing a novel gene editing therapeutic to target alveolar rhabdomyosarcoma. Alveolar rhabdomyosarcoma (aRMS) is a rare and aggressive soft tissue cancer arising from skeletal muscle stem cells in children and young adults. This project aims to leverage nanotechnology-based strategies to develop a lipid nanoparticle (LNP) platform designed to deliver CRISPR/Cas9 reagents configured to disrupt the PAX3-FOXO1 translocation that drives the most aggressive phenotypes of aRMS and mitigate its oncogenic effects. Here we present a two-pronged approach: first, designing and validating single guide RNA (sgRNA) sequences configured for the targeted knock out of PAX3-FOXO1 translocations; second, engineering LNPs for the efficient delivery of these gene-editing reagents for direct therapeutic use on aRMS tumors. PAX3-FOXO1 sgRNAs were designed using conserved fusion regions identified from ChimerDB 4.0 and the UCSC Genome database. We have been able to quantify consistent knockout of the PAX3-FOXO1 translocation mutation through delivery of LNPs in prior experiments via qPCR. We will be focusing this summer on quantitatively measuring the genotypic and phenotypic impacts of this knockout on an aRMS cell line (RH-30). This project aims to demonstrate the potential of LNP-based CRISPR/Cas9 strategies to target oncogenic mutations therapeutically in high-risk childhood malignancies like aRMS.