Author Identifier (ORCID)
Abstract
Despite the revolutionary impact of genome engineering tools in medicine, the safe and effective intracellular delivery of CRISPR remains a major obstacle to clinical applications. Here, we utilize precision molecular targeting and delivery strategies based on CRISPR-nuclease-dead Cas9 (dCas9) systems adapted for epigenetic repression (dCas9-Krüppel-associated box [KRAB]) to silence oncogenic drivers with high selectivity. As proof of principle, we target the EWSR1-FLI1 translocation, which encodes a chimeric and hard-to-drug oncogenic transcription factor driving approximately 85% of the cases of Ewing sarcoma (EWS)—an aggressive childhood malignancy. We describe the development of a programmable, non-viral polymeric system for the delivery of dCas9-KRAB as ribonucleoprotein (RNP) payloads for EWSR1-FLI1 repression. We demonstrate highly efficient intracellular delivery of RNPs loaded in polyamide-amine (PAMAM) polymers functionalized by guanidino groups, resulting in robust silencing of EWSR1-FLI1 both in established cell line xenografts and in EWS-related patient-derived xenografts (PDXs) of EWS. We show that silencing of EWSR1-FLI1 is accompanied by potent anti-tumor effects. Collectively, we characterize an effective non-viral platform for in vivo delivery of dCas9-KRAB/RNPs, which could be adapted for the repression of any oncogene. We further outline dCas9/RNP formulations for future therapeutic applications to treat poor-prognosis cancers driven by hard-to-drug oncogenes.
Keywords
CRISPR-dCas9, Ewing sarcoma, EWSR1-FLI1, genome engineering, oncogenes, polymeric nanoparticles, ribonucleoprotein complexes
Document Type
Journal Article
Date of Publication
1-1-2026
E-ISSN
15250024
ISSN
15250016
PubMed ID
42675859
Publication Title
Molecular Therapy
Publisher
Elsevier
School
Centre for Precision Health
Funding Information
This work was supported by a Cancer Council of Western Australia (CCWA) project grant (2004608) and the Research Excellence Award from the Department of Health (Western Australia) to P.B. S.T. would like to acknowledge the financial support of the Australian Government Research Training Program (RTP), Sock it to Sarcoma, and CCWA PhD Top Up Scholarships. C.W.E. acknowledges funding by the state government of Western Australia. R.L. was supported by an Australian National Health and Medical Research Council (NHMRC) Investigator Grant (GNT2035042). The PHS grant (CA165995) from the National Cancer Institute (USA) awarded to P.J.H. supported the establishment of the EWS model. The authors acknowledge Professor Vincent Rotello for providing the mCherry-E20 protein.
Funding received from the National Health and Medical Research Council (NHMRC)
GNT2035042
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.
Recommended Citation
Taifour, S., Wong, C., Wallis, C., Wang, E., Tie, A. W., Woodward, E., Waryah, C., Hosseinpour, M., Lam, D., Lister, R., Dymond, L., Woo, A., Houghton, P. J., Iyer, K. S., Norret, M., Evans, C. W., Winteringham, L., Gaudieri, S., & Blancafort, P. (2026). Targeting of the oncogenic fusion EWSR1-FLI1 in Ewing sarcoma by CRISPR/dCas9 silencers. Molecular Therapy. Advance online publication. https://doi.org/10.1016/j.ymthe.2026.08.042