P23

Optimising circular RNA production, expression efficiency, and delivery by viral pseudoparticles

V Arnaud(1,3,4,5,6,9) A Prel(1,3,4,5,6,9) M De la Pena(2,7) F Deschaseaux(1,3,4,5,6,9) J C Pagès(1,3,4,5,6,8,9)

1:Toulouse 3, Paul Sabatier; 2:Universidad Politécnica de Valencia-CSIC; 3:Inserm U1301; 4:CNRS UMR5070; 5:EFS-Occitanie; 6:ENVT; 7:Instituto de Biología Molecular y Celular de Plantas; 8:CHU de Toulouse, IFB, Hôpital Purpan; 9:RESTORE

RNA delivery is a promising approach to achieve rapid and transient gene expression, in oncology, cell and gene-based therapies as well as vaccination. However, most mRNAs are rapidly degraded by cellular exonucleases, which might limit their use. To counteract this, using circular RNAs (circRNA) could be a promising strategy since they seem to be more stable by resisting to exonuclease degradation, in addition, circRNA can be translated.

In this context, we develop new tools to circularize RNA and drive their packaging into pseudoparticles. For efficient delivery we use MS2-chimeric retrovirus-like particles. This system enables RNA encapsidation via a specific interaction between a phage protein (MS2-coat) and a RNA stem-loop (MS2 aptamer).

We constructed vectors containing hammerhead ribozymes (HHR) sequences at the 5’ and 3’ end of a cassette containing an IRES, a reporter gene and six stem-loop of MS2 RNA. The 5’ and 3’ HHR sequences enable self-cleavage generating 5’OH and 3’P end substrates of cellular ligase.

RNA circularization is confirmed by RT-PCR using divergent primers and junction sequencing. Expression of reporter gene were detected in transfected HEK293 cells. We observed differences in fluorescence intensity according to the type of circRNA constructions.

Besides HHR, CircRNA production will be improved thanks to the use of Tornado (Twister-optimized RNA for durable overexpression). Their encapsidation will be evaluated after constructs modifications.

Our ongoing work focuses on refining circRNA production, improving the translation efficiency of circRNA and of the delivery systems to enhance their functionality and therapeutic potential.