Mentor Name: Steven Jonas

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma primarily affecting children, adolescents, and young adults with especially poor outcomes for patients with metastatic solid tumors despite intensive chemotherapy, surgery, and radiation. Lipid nanoparticles (LNPs) are clinically validated delivery vehicles for gene editing and mRNA therapeutics, but lack of tumor cell targeting and inefficient penetration stand in the way of their implementation. This ALSF POST project seeks to engineer LNPs for RMS gene therapy by utilizing tumor-specific peptide targeting and examining stealth properties to improve tumor uptake and distribution. Aim 1 adapts the FGFR4-targeting peptide FYQ-8 as a targeting ligand to direct LNPs to RMS tumor cells. FGFR4 is commonly overexpressed in RMS, positioning it as an attractive tumor-associated receptor. Targeting peptides will be conjugated onto lipids via click chemistry, which will then be incorporated into LNP formulations. Aim 2 investigates how the density of PEG lipids affects their ability to penetrate RMS tumor organoids. PEGs are critical in LNP circulation and immune evasion, but when incorporated at very high densities, PEG can hinder endosomal escape, which is a process critically integral to payload release. Additionally, PEG keeps LNPs small and non-aggregating while lowering adhesiveness, optimal for tumors for penetration. LNPs with systematically varied PEG densities will be tested in 3D RMS organoid models to quantify penetration depth and transfection efficiency. By integrating receptor targeting ligands with rationally tuned PEG density, this work aims to design LNPs capable of selective homing and effective RMS tumor penetration, laying the groundwork for future therapy studies, including incorporation of acid-degradable PEG lipids.