Louisiana State University and Agricultural & Mechanical College

Biontech Ag

Uniwersytet Warszawski

Tron—translationale Onkologie An Der Universitátsmedizin Der Johannes Gutenberg—universitÄt Mainz Gemeinnützige Gmbh

University of Warsaw

Edward Darzynkiewicz

Jacek Jemielity

Joanna Kowalska

Robert E Rhoads

Ugur Sahin

Andreas Kuhn

Maciej Lukaszewicz

Joanna Zuberek

Ewa M Grudzien-Nogalska

Janusz Stepinski

The present invention relates to modification of RNA with 5'-cap analogs of Formula (1): wherein R-Rand n are as described herein, in order to improve the stability and increase the expression of said RNA, in particular in immature antigen presenting cells. The present invention provides a vaccine composition comprising said stabilized RNA, immature antigen presenting cells comprising said stabilized RNA, and methods for stimulating and/or activating immune effector cells and for inducing an immune response in an individual using said stabilized RNA.

Vaccine composition comprising 5'-cap modified RNA

Dinucleotide cap analogs are disclosed, modified at different phosphate positions with a boranophosphate group or a phosphoroselenoate group. The analogs are useful as reagents in the preparation of capped mRNAs and have increased stability both in vitro and in vivo. They may be used as inhibitors of cap-dependent translation. Optionally, the boranophosphate or phosphoroselenoate group has a 2'-O or 3&#x2032;-O-alkyl group, preferably a methyl group, producing analogs called BH-ARCAs or Se-ARCAs. ARCAs may be modified with &#x3b1;-, &#x3b2;-, or &#x3b3;-boranophosphate or phosphoroselenoate groups.

mRNA cap analogs

New RNA cap analogs are disclosed containing one or more phosphorothioates groups. The analogs also contain modifications at the 2'-O position of 7-methylguanosine that prevent them from being incorporated in the reverse orientation during in vitro synthesis of mRNA and that hence are 'anti-reverse cap analogs' (ARCAs). The ARCA modification ensures that the S atom is precisely positioned within the active sites of cap-binding proteins in both the translational and decapping machinery. The new S-ARCA analogs are resistant to in vivo decapping enzymes. Some S-ARCAs have a higher affinity for eIF4E than the corresponding analogs not containing a phosphorothioate group. When mRNAs containing the various S-ARCAs are introduced into cultured cells, some are translated as much as five-fold more efficiently than mRNAs synthesized with the conventional analog mGpppG.

Synthesis and use of anti-reverse phosphorothioate analogs of the messenger RNA cap

The ability to synthesize capped RNA transcripts in vitro has been of considerable value in a variety of applications. However, one-third to one-half of the caps have, until now, been incorporated in the reverse orientation. Such reverse caps impair the translation of in vitro-synthesized mRNAs. Novel cap analogues, such as P-3'-deoxy-7-methylguanosine-5&#x2032;P-guanosine-5&#x2032;triphosphate and P-3&#x2032;-O,7-dimethylguanosine-5&#x2032;P-guanosine-5&#x2032;triphosphate, have been designed that are incapable of being incorporated into RNA in the reverse orientation. Transcripts produced with SP6 polymerase using 'anti-reverse' cap analogues were of the predicted length. Analysis of the transcripts indicated that reverse caps were not formed. The in vitro translational efficiency of transcripts with the novel 'anti-reverse' cap analogues was significantly higher than that of transcripts formed with conventional caps.

Synthesis and use of anti-reverse mRNA cap analogues

Growing community of inventors

Warsaw, Poland

Edward Darzynkiewicz