Rna with a combination of unmodified and modified nucleotides for protein expression

Abstract

The invention relates to a polyribonucleotide with a sequence that codes a protein or protein fragment, wherein the polyribonucleotide comprises a combination of unmodified and modified nucleotides, wherein 5 to 50% of the uridine nucleotides and 5 to 50% of the cytidin nucleotides are modified uridine nucleotides or modified cytidin nucleotides.

1 .- 36 . (canceled) 
     
     
         37 . A method for producing erythropoietin in a mammal, comprising delivering to cells of the mammal an mRNA sequence encoding erythropoietin, wherein in the mRNA 15 to 30% of the uridine nucleotides are modified and 15 to 30% of the cytidine nucleotides are modified. 
     
     
         38 . The method of  claim 37 , wherein 15 to 25% of the uridine nucleotides are modified and 15 to 25% of the cytidine nucleotides are modified. 
     
     
         39 . The method of  claim 37 , wherein 15 to 30% of the uridine nucleotides are modified to 2-thiouridine (s2U) or 5-iodonridine (I5U). 
     
     
         40 . The method of  claim 37 , wherein 15 to 30% of the cytidine nucleotides are modified to 5-methylcytidine (m5C), 2′-amino-2′-deoxytidine (C2′NH2), or 2′-fluoro-2′-deoxycytidine (C2′F). 
     
     
         41 . The method of  claim 39 , wherein the uridine nucleotides are modified to 2-thioundine (s2U). 
     
     
         42 . The method of  claim 39 , wherein the uridine nucleotides are modified to 5-thioridine (I5U). 
     
     
         43 . The method of  claim 40 , wherein the cytidine nucleotides are modified to 5-methylcytidine (m5C). 
     
     
         44 . The method of  claim 40 , wherein the cytidine nucleotides are modified to 2′-fluoro-2′-deoxycytidine (C2′F′). 
     
     
         45 . The method of  claim 37 , wherein 25% of the unidine nucleotides are modified to 2-Thiouridine (s2U) and 25% of the cytidine nucleotides are modified to 5-methylcytidine (m5C). 
     
     
         46 . An mRNA encoding erythropoietin in which 15 to 30% of the uridine nucleotides are modified to 2-thiouridine (s2U) or 5-iodouridine (I5U) and 15 to 30% of the cytidine nucleotides are modified to 5-methylcytidine (m5C), 2′-amino-2′-deoxycytine (C2′NH2), or 2′-fluoro-2′-deoxycytidine (C2′F). 
     
     
         47 . The mRNA of  claim 46 , wherein 25% of the uridine nucleotides are modified and 25% of the cytidine nucleotides are modified. 
     
     
         48 . The mRNA of  claim 46 , further comprising a 5′ and a 3′ noncoding region flanking the sequence, and wherein the modified mRNA is formulated in a nanoparticle. 
     
     
         49 . The mRNA of  claim 46 , wherein the modified mRNA has a 5′ cap structure. 
     
     
         50 . The method of  claim 46 , wherein the modified mRNA has a polyA tail. 
     
     
         51 . A kit for increasing hematocrit of a mammal, comprising a sterile container comprising the mRNA of  claim 46 . 
     
     
         52 . A method of increasing hematocrit of a mammal in need thereof, comprising administering to the mammal an mRNA encoding erythropoietin in which 15 to 30% of the uridine nucleotides are modified to 2-thiouridine (s2U) or 5-iodouridine (I5U) and 15 to 30% of the cytidine nucleotides are modified to 5-methylcytidine (m5C) 2′-amino-2′-deoxycytidine (C2′NH2), or 2′-fluoro-2′-deoxycytidine (C2′F), wherein said mRNA is effective in increasing the haematocrit in the mammal. 
     
     
         53 . The method of  claim 52 , wherein said administration is repeated.