Abstract Ewing sarcoma is a high-risk sarcoma with overall 50% survival, unchanged for decades. No effective new therapies have been introduced in that time frame. It is thus an ideal candidate for targeted therapy. It is also a tumor with several candidate targets for therapy. Since essentially all Ewing sarcomas express the cell surface antigen CD99, monoclonal antibodies can be used to deliver nanoparticle-encapsulated chemotherapeutic agents with high efficiency. Compared to conventional antibody-drug conjugates (ADCs), binding avidity and delivered dose per bound antibody is increased exponentially. Similarly, the tumor-specific EWS-FLI1 or equivalent gene translocation that drives oncogenesis is an attractive candidate target, so far untargetable with conventional small molecules. Both nanoparticle-delivered shRNA and CRISPR/Cas9 constructs targeting the fusion gene are candidate therapeutics that can be directed specifically to the fusion junction or the ets domain. Downstream targets of the fusion gene include a number of coding and noncoding genes, among them FEZF1-AS, a long noncoding RNA (lncRNA) that we have shown is both a direct target of the Ewing fusion gene and an oncogenic driver of tumor growth, invasion, and metastasis. While otherwise untargetable, lncRNAs can be effectively downregulated by ASOs (anti-sense oligonucleotides) with a proportional attenuation of their oncogenic effect. Here we present evidence that a novel CD99 targetable nanoparticle, termed hybrid polymerized liposomal nanoparticles, can effectively deliver small-molecule cytotoxics with complete tumor ablation in a nude mouse model of Ewing sarcoma, as well as EWS-FLI1 targeted CRISPR/Cas9 constructs and FEZF1-AS targeted ASOs, with proportional knockdown and oncogenic attenuation. Tumor uptake of the therapeutic nanoparticles is rapid, with over 90% of particles at therapeutic doses bound and internalized within 20 minutes in vitro. The IC50 for cytotoxics is improved 40-fold over free agent and 20-fold over untargeted nanoparticle formulations. Animals with Ewing sarcoma xenografts treated with CD99-targeted hPLNs containing irinotecan show complete tumor ablation with no tumor regrowth at 90 days, 20 days after cessation of treatment, unlike free irinotecan or untargeted irinotecan liposomal nanoparticle animals, all of whom died with progressive tumor growth. Surviving animals showed no toxicity. Ancillary studies identified no evidence of bone marrow, liver, or kidney toxicity at therapeutic doses. Over 70% of tumor cells express EGFP after internalizing targeted nanoparticles containing CRISPR/Cas9 plasmids with an EGFP construct. Similarly, FEZF1-AS lncRNA expression is reduced by over 70% after treatment with FEZF1-AS specific ASOs. Taken together, these results suggest that both targetable (cytotoxics delivered preferentially to tumor cells) and previously untargetable features (fusion genes, lncRNAs) can be effectively and specifically treated when conventional and novel therapeutics are delivered directly to tumor cells by CD99-targeted nanoparticles. The successful treatment of Ewing sarcoma xenografts and metastases in animal models by any of these approaches also suggests combined therapy with both conventional and unconventional agents is feasible, with no appreciable toxicity. Citation Format: Timothy J. Triche, Jon Nagy, Hyung Kang, Chris Denny, Sheetal Bajaj Mitra. Targeted therapy of Ewing sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A28.