title: mRNA transfection of dendritic cells: synergistic effect of ARCA mRNA capping with Poly(A) chains in cis and in trans for a high protein expression level.
abstract: The occurrence of translation mechanism in the cytosol offers advantages to mRNA transfer over DNA-based transfection in non-dividing cells. Here, we sought to optimize mRNA constructs allowing a high level of protein upon lipofection. We found that luciferase into mouse dendritic cells (JAWSII cells) was approximately 20-fold higher when the luciferase mRNA was capped with 3'-O-methyl-m7(5')Gppp5'G (anti-reverse cap analogue; ARCA) than with m7(5')Gppp5'G (CAP). Adding a Poly(A) tail of 100 instead of 64 adenosines in cis increased by approximately 35-fold more the protein level. Finally, ARCA-Luc-mRNA-A100 construct was 700-fold better efficient than the CAP-Luc-mRNA-A64 one. Moreover, co-transfection with free Poly(A) chains in trans enhanced by 100% the luciferase level. The efficiency of ARCA-mRNA-A100 construct was validated in immature and mature human CD34-derived dendritic cells. Such mRNA construct was also successful to obtain high level of MART-1 tumor antigen.
authors.affiliation: Centre de Biophysique Moléculaire, UPR4301 CNRS, affiliated to the University of Orléans and INSERM, rue Charles Sadron, 45071 Orléans cedex 2, France.
authors.forename: Michael
authors.initials: M
authors.lastname: Mockey
authors.forename: Cristine
authors.initials: C
authors.lastname: Gonçalves
authors.forename: Franck P
authors.initials: FP
authors.lastname: Dupuy
authors.forename: François M
authors.initials: FM
authors.lastname: Lemoine
authors.forename: Chantal
authors.initials: C
authors.lastname: Pichon
authors.forename: Patrick
authors.initials: P
authors.lastname: Midoux
mesh_terms: D000818:Animals; D002460:Cell Line; D003713:Dendritic Cells; D051379:Mice; D015202:Protein Engineering / Q000379:methods*; D012314:RNA Cap Analogs / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection / Q000379:methods*
chemical_list: D012314:RNA Cap Analogs; D012333:RNA, Messenger; D011994:Recombinant Proteins