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 cytidine nucleotides are modified uridine nucleotides or modified cytidine nucleotides.

1 - 40 . (canceled) 
     
     
         41 . A therapeutic agent comprising a modified polyribonucleotide having a sequence which encodes a protein or functional fragment thereof, wherein said modified polyribonucleotide comprises a combination of unmodified and modified nucleotides, which modified nucleotides comprise analogs of uridine or analogs of cytidine, wherein:
 (a) said modified polyribonucleotide induces less than a 5-fold change in a level of at least one inflammatory marker selected from TNF-α, IL-6, IL-8, IL-12, IFN-α, IFN-3 and IFN-γ expressed by peripheral blood mononuclear cells exposed to said modified polyribonucleotide as compared to a level of said at least one inflammatory marker in peripheral blood mononuclear cells in a control that has been exposed to an unmodified polyribonucleotide encoding said protein or functional fragment thereof; and   (b) said modified polyribonucleotide provides an amount of said protein or functional fragment thereof over a time period in cells having said modified polyribonucleotide, wherein said time period is up to 4 weeks, and wherein said amount is increased as compared to an amount in control cells having said unmodified polyribonucleotide, as determined by a percentage content of red fluorescent protein in said cells when said protein or functional fragment thereof is red fluorescent protein.   
     
     
         42 . The therapeutic agent of  claim 41 , wherein said modified polyribonucleotide is formulated in a nanoparticle or nanocapsule. 
     
     
         43 . The therapeutic agent of  claim 41 , wherein said modified polyribonucleotide is formulated in a cationic lipid, cationic polymer, or nanoemulsion. 
     
     
         44 . The therapeutic agent of  claim 41 , wherein said modified polyribonucleotide comprises (i) uridine and cytidine; and (ii) analogs of said uridine and cytidine. 
     
     
         45 . The therapeutic agent of  claim 44 , wherein said modified polyribonucleotide comprises uridines and cytidines, and 5% to 50% of the uridines are analogs of uridine and 5% to 50% of the cytidines are analogs of cytidine. 
     
     
         46 . The therapeutic agent of  claim 45 , wherein said modified polyribonucleotide comprises uridines and cytidines, and 15% to 30% of the uridines are analogs of uridine and 15% to 30% of the cytidines are analogs of cytidine. 
     
     
         47 . The therapeutic agent of  claim 44 , wherein said analogs of uridine are selected from the group consisting of 2-thiouridine, 5-iodouridine, and 5-methyluridine. 
     
     
         48 . The therapeutic agent of  claim 44 , wherein the modified polyribonucleotide does not comprise pseudouridine. 
     
     
         49 . The therapeutic agent of  claim 44 , wherein said analogs of cytidine are selected from the group consisting of 5-methylcytidine, 2′-amino-2′-deoxycytidine, 2′-fluoro-2′-deoxycytidine, and 5-iodocytidine. 
     
     
         50 . The therapeutic agent of  claim 44 , wherein said modified polyribonucleotide comprises 5-methylcytidine. 
     
     
         51 . The therapeutic agent of  claim 41 , wherein said modified polyribonucleotide comprises (i) adenosine or guanosine; and (ii) analogs of said adenosine or guanosine. 
     
     
         52 . The therapeutic agent of  claim 41 , wherein said modified polyribonucleotide comprises less than 20% analogs of adenosine or guanosine. 
     
     
         53 . The therapeutic agent of  claim 52 , wherein said modified polyribonucleotide does not comprise analogs of adenosine or analogs of guanosine. 
     
     
         54 . The therapeutic agent of  claim 41 , wherein said modified polyribonucleotide has a transfection efficiency greater than 80% among cells exposed to said modified polyribonucleotide. 
     
     
         55 . The therapeutic agent of  claim 41 , wherein said at least one inflammatory marker comprises at least two of TNF-α, IL-6, IL-8, IL-12, IFN-α, IFN-β and IFN-γ. 
     
     
         56 . The therapeutic agent of  claim 41 , wherein said modified polyribonucleotide comprises a 3′ or 5′ noncoding region flanking said sequence which encodes said protein or functional fragment thereof, wherein said noncoding region aids in said amount of said protein or functional fragment thereof in said cells for said time period. 
     
     
         57 . The therapeutic agent of  claim 41 , wherein said modified polyribonucleotide induces substantially no change in said level of said at least one inflammatory marker expressed by peripheral blood mononuclear cells exposed to said modified polyribonucleotide. 
     
     
         58 . The therapeutic agent of  claim 41 , wherein (i) said sequence is a gene or fragment whose defect or deficiency is associated with a presence of a disease, or (ii) a lack or deficiency of said protein or functional fragment is associated with the presence of said disease. 
     
     
         59 . The therapeutic agent of  claim 41 , wherein said time period is up to 5 days. 
     
     
         60 . A therapeutic agent comprising a modified polyribonucleotide having a sequence which encodes a protein or functional fragment thereof, wherein said modified polyribonucleotide comprises a combination of unmodified and modified nucleotides, which modified nucleotides comprise analogs of uridine or analogs of cytidine, wherein:
 (a) said modified polyribonucleotide lowers an immune response of a subject by at least 30% as compared to an unmodified polyribonucleotide encoding said protein or functional fragment thereof, which immune response is as determined by a level of at least one inflammatory marker selected from TNF-α, IL-6, IL-8, IL-12, IFN-α, IFN-β and IFN-γ expressed by peripheral blood mononuclear cells exposed to said modified polyribonucleotide as compared to a level of said at least one inflammatory marker in peripheral blood mononuclear cells in a control that has been exposed to an unmodified polyribonucleotide encoding said protein or functional fragment thereof; and   (b) said modified polyribonucleotide provides an amount of said protein or functional fragment thereof over a time period in cells of said subject having said modified polyribonucleotide, wherein said time period is up to 4 weeks, and wherein said amount is increased as compared to an amount in control cells having said unmodified polyribonucleotide, as determined by a percentage content of red fluorescent protein in said cells when said protein or functional fragment thereof is red fluorescent protein.   
     
     
         61 . The therapeutic agent of  claim 60 , wherein said modified polyribonucleotide comprises (i) uridine and cytidine; and (ii) analogs of said uridine and cytidine. 
     
     
         62 . The therapeutic agent of  claim 61 , wherein said modified polyribonucleotide comprises uridines and cytidines, wherein 5% to 50% of the uridines are analogs of uridine and 5% to 50% of the cytidines are analogs of cytidine. 
     
     
         63 . The therapeutic agent of  claim 62 , wherein said modified polyribonucleotide comprises uridines and cytidines, wherein 15% to 30% of the uridines are analogs of uridine and 15% to 30% of the cytidines are analogs of cytidine. 
     
     
         64 . The therapeutic agent of  claim 61 , wherein said modified polyribonucleotide comprises 5-methylcytidine. 
     
     
         65 . The therapeutic agent of  claim 60 , wherein said modified polyribonucleotide comprises less than 20% analogs of adenosine or guanosine. 
     
     
         66 . The therapeutic agent of  claim 60 , wherein said modified polyribonucleotide lowers an immune response of said subject by at least 50% as compared to said unmodified polyribonucleotide. 
     
     
         67 . The therapeutic agent of  claim 60 , wherein said modified polyribonucleotide comprises a 3′ or 5′ noncoding region flanking said sequence which encodes said protein or functional fragment thereof, wherein said noncoding region aids in said amount of said protein or functional fragment thereof in said cells for said time period. 
     
     
         68 . The therapeutic agent of  claim 60 , wherein (i) said sequence is a gene or fragment whose defect or deficiency is associated with a presence of a disease, or (ii) a lack or deficiency of said protein or functional fragment is associated with the presence of said disease. 
     
     
         69 . The therapeutic agent of  claim 60 , wherein said time period is up to 5 days.