P77

Therapeutic extracellular vesicles (TEVs) to delivery Cas9-sgRNA complex for Ewing sarcoma treatment

S T Cervera Mayor(1,2) R Melero Fernández de Mera(1,2) S Martínez Rodriguez(1) C Rodriguez Martín(1,2) E Fernandez Tabanera(1,2) S Josa(1) J Alonso(1,2)

1:Instituto de Salud Carlos III; 2:CIBERER

Ewing sarcoma is a rare and aggressive bone cancer affecting children and young adults. These tumours are characterised by chromosomal translocations that results in chimeric  transcription factors (i.e. EWSR1-FLI1) that govern the tumorigenesis process. Previous studies carried out in our group have demonstrated that EWSR1-FLI1 gene inactivation using CRISPR-Cas9 tools produced cell growth arrest and senescence. However, delivery of Cas9 machinery to target cells in vivo is challenge and new approaches are necessary. Extracellular vesicles for Cas9 ribonucleoprotein delivery has emerged as an interesting tool due to its low immunogenicity and versatility. We have generated therapeutic extracellular vesicles (TEVs) containing Cas9 and specific sgRNAs designed to inactive EWSR1-FLI1 using a combination of molecular strategies that include the use of RNA binding proteins, ribozymes and protein dimerization domains. TEVs could be isolated from the culture medium of HEK293T cells transfected with the appropriate plasmids. Cas9 amount contain in TEVs was quantified by western blot using a Cas9 recombinant protein standard curve. In vitro assays showed that the addition of TEVs to the Ewing sarcoma cell line A673 produced around 50% gene editing at EWSR1-FLI1 gene after 72h demonstrating the effectiveness of this approach. We are improving this delivery system to increase their specificity to target Ewing sarcoma cells for its application in vivo. In conclusion, the use of TEVs to induce gene editing at least in vitro is efficient and would be readily applicable to other pathologies in which gene editing-based therapies could represent a therapeutic option.