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 .- 30 . (canceled) 
     
     
         31 . A method for producing erythropoetin 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. 
     
     
         32 . The method of  claim 31 , wherein 15 to 25% of the uridine nucleotides are modified and 15 to 25% of the cytidine nucleotides are modified. 
     
     
         33 . The method of  claim 31 , wherein 15 to 30% of the uridine nucleotides are modified to 2-thiouridine (s2U) or 5-iodouridine (I5U). 
     
     
         34 . The method of  claim 31 , wherein 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). 
     
     
         35 . The method of  claim 33 , wherein the uridine nucleotides are modified to 2-thiouridine (s2U). 
     
     
         36 . The method of  claim 33 , wherein the uridine nucleotides are modified to 5-iodouridine (15U). 
     
     
         37 . The method of  claim 34 , wherein the cytidine nucleotides are modified to 5-methylcytidine (m5C). 
     
     
         38 . The method of  claim 34 , wherein the cytidine nucleotides are modified to 2′-fluoro-2′-deoxycytidine (C2′F). 
     
     
         39 . The method of  claim 31 , wherein 25% of the uridine nucleotides are modified to 2-thiouridine (s2U) and 25% of the cytidine nucleotides are modified to 5-methylcytidine (m5C). 
     
     
         40 . An mRNA encoding erythropoietin in which 15 to 30% of the uridine nucleotides are modified to 2-thiouridine (s2U) or 5-iodouridine (15U) 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). 
     
     
         41 . The mRNA of  claim 40 , wherein 25% of the uridine nucleotides are modified and 25% of the cytidine nucleotides are modified. 
     
     
         42 . The mRNA of  claim 40 , further comprising a 5′ and a 3′ noncoding region flanking the sequence, and wherein the modified mRNA is formulated in a nanoparticle. 
     
     
         43 . The mRNA of  claim 40 , wherein the modified mRNA has a 5′ cap structure. 
     
     
         44 . The method of  claim 40 , wherein the modified mRNA has a polyA tail. 
     
     
         45 . A kit for increasing hematocrit of a mammal, comprising a sterile container comprising the mRNA of  claim 40 . 
     
     
         46 . 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. 
     
     
         47 . The method of  claim 46 , wherein said administration is repeated.