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 nucleotide, 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 polyribonucleotide comprising a sequence which encodes a protein or protein fragment, wherein the polyribonucleotide contains a combination of unmodified and modified nucleotides, wherein 5 to 50% of uridine nucleotides in the polyribonucleotide are 2-thiouridine, and 5 to 50% of cytidine nucleotides in the polyribonucleotide are 5-methylcytidine. 
     
     
         32 . The polyribonucleotide of  claim 31 , further comprising a micro-RNA binding site. 
     
     
         33 . The polyribonucleotide of  claim 31 , wherein the polyribonucleotide includes a 3′ poly A tail, and wherein the micro-RNA binding site is 3′ to the sequence which encodes a protein or protein fragment and 5′ from the poly A tail. 
     
     
         34 . The polyribonucleotide of  claim 31 , wherein the polyribonucleotide is mRNA. 
     
     
         35 . The polyribonucleotide of  claim 31 , wherein 5 to 30% of uridine nucleotides in the polyribonucleotide are 2-thiouridine and 5 to 30% of cytidine nucleotides in the polyribonucleotide are 5-methylcytidine. 
     
     
         36 . The polyribonucleotide of  claim 31 , wherein 7.5 to 25% of uridine nucleotides in the polyribonucleotide are 2-thiouridine and 7.5 to 25% of cytidine nucleotides in the polyribonucleotide are 5-methylcytidine. 
     
     
         37 . The polyribonucleotide of  claim 31 , wherein the polyribonucleotide contains an RNA sequence encoding a growth hormone, cystic fibrosis transmembrane conductance regulator (CFTR), a growth factor, an angiogenesis factor, a stimulator, an inducer, an enzyme, a T cell receptor, erythropoietine (EPO), BMP-2 or a fragment thereof. 
     
     
         38 . The polyribonucleotide of  claim 31 , wherein the polyribonucleotide contains an RNA sequence encoding SP-B or ABCA3. 
     
     
         39 . The polyribonucleotide of  claim 31 , wherein the polyribonucleotide contains an RNA sequence encoding CFTR. 
     
     
         40 . The polyribonucleotide of  claim 31 , wherein the polyribonucleotide can express tailored T cell receptors. 
     
     
         41 . The polyribonucleotide of  claim 31 , wherein the polyribonucleotide contains a sequence encoding an antigenic part of a pathogen. 
     
     
         42 . The polyribonucleotide of  claim 31 , wherein the polyribonucleotide contains a sequence encoding EPO. 
     
     
         43 . The polyribonucleotide of  claim 31 , wherein the polyribonucleotide contains at least one sequence encoding a growth factor, an angiogenesis factor, a stimulator, an inducer or an enzyme. 
     
     
         44 . A composition comprising the polyribonucleotide of  claim 31 . 
     
     
         45 . The composition of  claim 44 , wherein the polyribonucleotide is formulated in a nanoparticle or nanocapsule. 
     
     
         46 . The composition of  claim 44 , wherein the polyribonucleotide is formulated in a cationic lipid, cationic polymer, or nanoemulsion. 
     
     
         47 . The composition of  claim 44 , wherein 5 to 30% of uridine nucleotides in the polyribonucleotide are 2-thiouridine and 5 to 30% of cytidine nucleotides in the polyribonucleotide are 5-methylcytidine. 
     
     
         48 . The composition of  claim 44 , wherein the composition is formulated for intratracheal and/or pulmonary administration. 
     
     
         49 . A method of decreasing binding of a polyribonucleotide to retinoic acid-inducible gene I (RIG-1), comprising
 producing a polyribonucleotide comprising a sequence which encodes a protein or protein fragment, wherein the polyribonucleotide contains a combination of unmodified and modified nucleotides, wherein 5 to 50% of uridine nucleotides in the polyribonucleotide are 2-thiouridine, and 5 to 50% of cytidine nucleotides in the polyribonucleotide are 5-methylcytidine;   providing the polyribonucleotide so produced; and   administering the polyribonucleotide to a subject or cell, wherein the protein or protein fragment encoded by the polyribonucleotide is expressed in the subject or cell, and wherein the polyribonucleotide has decreased binding to RIG-1 relative to a control polyribonucleotide that does not comprise the analogs.   
     
     
         50 . The method of  claim 49 , wherein the remainder of the ATP, CTP, GTP and UTP in the polyribonucleotide does not include a modified nucleoside. 
     
     
         51 . The method of  claim 49 , further comprising encapsulating said polyribonucleotide in a nanoparticle or nanocapsule. 
     
     
         52 . The method of  claim 49 , further comprising encapsulating said polyribonucleotide in a cationic lipid, cationic polymer, or nanoemulsion. 
     
     
         53 . The method of  claim 49 , wherein 5 to 30% of uridine nucleotides in the polyribonucleotide are 2-thiouridine and 5 to 30% of cytidine nucleotides in the polyribonucleotide are 5-methylcytidine