Click-modified mrna

Abstract

The present invention relates to alkyne- and/or azide-modified mRNA, processes for producing such modified mRNA, cells which are transfected to include the modified mRNA, pharmaceutical compositions containing the modified mRNA or cells including the modified mRNA, and to uses of such mRNA, cells or pharmaceutical compositions in mRNA based therapeutic and/or prophylactic applications.

1 . Modified messenger RNA (mRNA), comprising a 5′-cap structure, a 5′-untranslated region (5′-UTR), an open reading frame region (ORF), a 3′-untranslated region (3′-UTR) and a poly(A) tail region, characterized in that it contains at least one of an alkyne- or azide-modification in at least one nucleotide within at least one of the ORF, the 5′-UTR, the 3′-UTR and the poly(A) tail region. 
     
     
         2 . Modified mRNA according to  claim 1 , characterized in that it contains modified nucleotides in
 a) the ORF and the UTRs,   b) the ORF, the UTRs and the poly(A) tail, or   c) only the poly(A) tail.   
     
     
         3 . Modified mRNA according to  claim 1  or  2 , wherein at least one of the four standard types of nucleotides (AMP, CMP, GMP, UMP) are partly or completely modified, preferably ethynyl- or azido-modified at uracil or adenine. 
     
     
         4 . Modified mRNA according to anyone of  claims 1  to  3 , wherein at least one nucleotide is alkyne-modified and at least one nucleotide is azide-modified. 
     
     
         5 . Modified mRNA according to any one of the preceding claims, wherein at least one of the four standard types of nucleotides is present in modified form compared to the non-modified form in a ratio of 1:100 to 10:1, preferably 1:10 to 1:10 or 1:1. 
     
     
         6 . Modified mRNA according to any one of the preceding claims, characterized in that it contains otherwise modified natural or artificial nucleotides, preferably pseudouridine or N1-methylpseudouridine. 
     
     
         7 . Modified mRNA according to any one of the preceding claims, wherein the modified mRNA contains one or more of a detectable label and a functional molecule introduced via a click reaction of the modified mRNA with a correspondingly modified alkyne- or azide-containing detectable label or functional molecule. 
     
     
         8 . Modified mRNA according to  claim 7 , wherein the detectable label is a colored or fluorogenic molecule and/or the functional molecule is a tissue or cell specific targeting group or ligand, preferably a sugar moiety or a fatty acid moiety. 
     
     
         9 . Modified RNA containing at least one alkyne- or azide-modification in at least one nucleotide or modified mRNA according to anyone of  claims 1  to  8 , which is complexed with a cationic or polycationic compound. 
     
     
         10 . Process for the production of the modified mRNA according to any one of the preceding claims, wherein the process comprises in vitro transcribing mRNA from a DNA template or alternatively performing a fermentation process using prokaryotic or eukaryotic host cells to express a DNA template contained in an expression vector wherein the process is performed in the presence of an RNA polymerase and a nucleotide mixture containing the four standard types of nucleotides required for mRNA transcription, in which nucleotide mixture at least a part of at least one of the four types of nucleotides is modified to contain an alkyne- or azide-modification. 
     
     
         11 . Process for the production of a modified mRNA containing an alkyne- or an azide-modification at the poly(A) tail, wherein the process comprises performing a poly(A) polymerase addition reaction at the poly(A) tail on an mRNA in the presence of ATP, wherein ATP is at least partly alkyne- or azide-modified at the adenosine. 
     
     
         12 . Process according to  claim 10  or  11 , further comprising adding a correspondingly alkyne- or azide-modified detectable label and/or functional molecule under conditions to perform a click reaction to produce a modified mRNA according to  claim 7  or  8 . 
     
     
         13 . Cell, which is obtained by ex vivo transfection of a corresponding human, animal or plant parent cell with a modified mRNA according to anyone of  claims 1  to  9 . 
     
     
         14 . Cell according to  claim 13 , wherein the cell is a cell of the human or animal immune system. 
     
     
         15 . Pharmaceutical composition, comprising a modified mRNA according to anyone of  claims 1  to  9  ora cell according to  claim 13  or  14  as an active agent, optionally in combination with a pharmaceutically acceptable adjuvant or excipient and/or contained in pharmaceutically acceptable carrier. 
     
     
         16 . Modified mRNA according to anyone of  claims 1  to  9  or a pharmaceutical composition according to  claim 15  for use in mRNA based therapeutic and/or prophylactic applications. 
     
     
         17 . Modified mRNA according to anyone of  claims 1  to  9  or pharmaceutical composition according to  claim 15 , especially use in therapeutic and/or prophylactic application according to  claim 16 , wherein the therapeutic and/or prophylactic application comprises targeted delivery in gene replacement therapy, targeted gene therapy in combination with specific endonucleases encoded by the mRNA (e.g. CRISPR/Cas9), in vaccination, in cancer therapy and for cell specific gene expression or gene editing for treatment of (inherited) diseases and genetic aberrations, or the use as an immunological adjuvant. 
     
     
         18 . Modified mRNA according to anyone of  claims 1  to  9  or pharmaceutical composition according to  claim 15 , especially for use in therapeutic and/or prophylactic application according to  claim 16  or  17  in a human or an animal. 
     
     
         19 . Use of a modified mRNA according to anyone of  claims 1  to  9  for transfection of plants and plant cells. 
     
     
         20 . A kit for production and/or delivery of a modified mRNA according to anyone of  claims 1  to  9 . 
     
     
         21 . Diagnostic composition for the in vitro screening for the presence, delivery and/or distribution of the modified mRNA according to anyone of  claims 1  to  9  in cells, tissue or organs, wherein the composition contains an mRNA including a detectable label, preferably a fluorophore label or a radionuclide label. 
     
     
         22 . Diagnostic composition for use in in vivo screening for the presence, delivery and/or distribution of the modified mRNA according to anyone  claims 1  to  9  or for in vivo monitoring of the re-administration of a cell according to  claim 13  or  14 , wherein the composition contains a modified mRNA including a fluorophore label or a radionuclide label, or the cell is transfected by a modified mRNA including a fluorophore label or a radionuclide label. 
     
     
         23 . Method for stabilizing RNA, especially mRNA, wherein an alkyne- and/or an azide-modification is introduced by including at least one of the four standard types of nucleotides (ATP, CTP, GTP and UTP) in partly or completely alkyne- and/or azide-modified form during RNA synthesis and/or in a poly(A) polymerase addition reaction to produce a modified (m)RNA, and optionally one or more of a detectable label and a functional molecule are introduced via a click reaction of the modified (m)RNA with a correspondingly modified alkyne- or azide-containing detectable label or functional molecule. 
     
     
         24 . In vitro method for qualitatively or quantitatively determining delivery and transfection of a modified mRNA according to any one of  claims 1  to  9  to target cells via a fluorescence-activated cell scanning analysis, which modified mRNA contains one or more fluorogenic molecules introduced via a click reaction to the modified mRNA with a correspondingly modified alkyne- or azide-containing fluorogenic molecule and/or which modified mRNA encodes a fluorescent protein. 
     
     
         25 . In vitro-method according to  claim 24 , wherein the fluorescence signals emitted by the fluorogenic molecule or the fluorescent protein are determined for target cells transfected with the modified mRNA and compared to non-transfected target cells.