MODIFIED RNA FOR INCREASING PROTEIN EXPRESSION
Described herein are modified RNA molecules where a 3′-stabilizing region is covalently attached to the RNA, and where the 3′-stabilizing region comprises one or more modified nucleosides. Methods of synthesizing said RNAs are also provided herein.
1 - 69 . (canceled)
70 . An RNA molecule comprising the structure of Formula I:
A−B (Formula I)
wherein A comprises:
a) a 5′-cap;
b) an open reading frame (ORF) encoding a protein; and
c) a poly-A region, wherein the poly-A region is 3′ to the open reading frame; and
B comprises a 3′-stabilizing region comprising 1 to 50 nucleosides, wherein one or more nucleosides within the 3′-stabilizing region comprise one or more purification handles.
71 . The RNA molecule of claim 70 , wherein the 3′-stabilizing region is covalently linked to a precursor RNA by a linker that can be formed by ligation.
72 . The RNA molecule of claim 70 , wherein the 3′-stabilizing region is covalently linked to a precursor RNA by a linker that can be formed using a polymerase.
73 . The RNA molecule of claim 70 , wherein the purification handle is linked to the 3′-stabilizing region via a linker (L).
74 . The RNA molecule of claim 70 , wherein the purification handle comprises a lipid.
75 . An RNA molecule comprising the structure of Formula II:
A-B-L (Formula II)
wherein A comprises:
a) a 5′-cap;
b) an open reading frame (ORF) encoding a protein; and
c) a poly-A region, wherein the poly-A region is 3′ to the open reading frame; and
B comprises a 3′-stabilizing region comprising 1 to 50 nucleosides, wherein one or more nucleosides within the 3′-stabilizing region comprise one or more linkers (L), wherein the linker (L) is capable of binding a purification handle.
76 . The RNA molecule of claim 75 , wherein the 3′-stabilizing region is covalently linked to a precursor RNA by a linker that can be formed by ligation.
77 . The RNA molecule of claim 76 , wherein the 3′-stabilizing region is covalently linked to a precursor RNA using a polymerase.
78 . The RNA molecule of claim 75 , wherein one or more purification handles are covalently linked to one or more nucleosides or one or more linkers within the 3′-stabilizing region.
79 . The RNA molecule of claim 70 , wherein the purification handle comprises a lipid.
80 . The RNA molecule of claim 70 , wherein the 3′-stabilizing region forms a secondary structure, optionally wherein the secondary structure is a hairpin loop.
81 . The RNA molecule of claim 70 , wherein the 3′-stabilizing region comprises one or more unmodified nucleosides and one or more unmodified internucleotide linkages.
82 . The RNA molecule of claim 70 , wherein the 3′-stabilizing region comprises one or more modified nucleosides and/or one or more modified internucleotide linkages.
83 . The RNA molecule of claim 82 , wherein the modified nucleoside comprises a modified nucleobase and/or a modified sugar.
84 . The RNA molecule of claim 82 , wherein the modified nucleoside comprises a modified nucleobase and the modified nucleobase is
(a) a modified uracil, a modified cytosine, a modified guanine, or a modified adenine;
(b) pseudouracil (y), 2-thio-uracil, 4-thio-uracil, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil, 5-halo-uracil, 3-methyl-uracil, 5-aza-uracil, or 2-thio-5-aza-uracil;
(c) 5-aza-cytosine, 6-aza-cytosine, pseudoisocytidine, 3-methyl-cytosine, 5-methyl-cytosine, 5-halo-cytosine, 2-thio-cytosine, or 2-thio-5-methyl-cytosine;
(d) 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, 2-amino-6-methyl-1-purine, 8-azido-adenine, 7-deaza-adenine, N6-methyl-adenine, or 2-methylthio-N6-methyl-adenine; or
(e) inosine, 1-methyl-inosine, 7-cyano-7-deaza-guanine, 7-aminomethyl-7-deaza-guanine, 6-thio-1-guanine, 6-thio-7-deaza-guanine, or 6-methoxy-guanine.
85 . The RNA molecule of claim 82 , wherein the modified nucleoside comprises a modified sugar and the modified sugar has a 5-membered ring or a 6-membered ring, or is a modified ribose, wherein the modified ribose is 2′-thioribose, 2′, 3′-dideoxyribose, 2′-amino-2′-deoxyribose, 2′ deoxyribose, 2′-azido-2′-deoxyribose, 2′-fluoro-2′-deoxyribose, 2′-O-methylribose, 2′-O-methyldeoxyribose, or 3′-amino-2′,3′-dideoxyribose.
86 . The RNA molecule of claim 82 , wherein the modified nucleoside comprises a morpholino ring, or wherein the internucleotide linkage comprises a modified phosphate.
87 . The RNA molecule of claim 86 , wherein (i) the internucleotide linkage comprises a modified phosphate and the modified phosphate is phosphorothioate, phosphorodithioate, thiophosphate, 5′-O-methylphosphonate, 3′-O-methylphosphonate, 5′-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate, methylphosphonate, or guanidinopropyl phosphoramidate; and/or (ii) the last nucleoside of the 3′-stabilizing region is ddC, inverted dT, 3′-phosphate nucleoside, 3′-oxime nucleoside, 3′-azidomethyl nucleoside, or 3′-methyl nucleoside.
88 . The RNA molecule of claim 70 , wherein the poly-A region is 10 or greater nucleosides in length.
89 . The RNA molecule of claim 70 , wherein the poly-A region is from 15 to 150 nucleosides in length.
90 . The RNA molecule claim 70 , wherein the RNA molecule is a messenger RNA (mRNA).
91 . A cell comprising the RNA molecule of claim 70 , optionally wherein the cell is isolated.
92 . A pharmaceutical composition comprising the RNA molecule, or comprising a cell comprising the RNA molecule, of claim 70 and a pharmaceutically acceptable carrier.
93 . A method of increasing the expression of a protein or a peptide of interest in a cell, comprising contacting the cell with the RNA molecule of claim 70 , wherein the RNA molecule encodes the protein or peptide of interest
(a) wherein the expression is increased when compared to that of an RNA molecule without the 3′-stabilizing region;
(b) wherein the cell translates the protein or peptide of interest from the RNA molecule; or
(c) wherein the half-life is increased when compared to that of an RNA molecule without the 3′-stabilizing region,
optionally wherein the cell is isolated, in vitro, or ex vivo.
94 . A method of preparing the RNA molecule of claim 70 , comprising covalently joining a stabilizing region to a precursor RNA comprising a 5′-cap, an ORF encoding a protein, and a poly-A region 3′ of the ORF, wherein the stabilizing region is added 3′ to the poly-A region and the stabilizing region comprises one or more purification handles and/or one or more linkers capable of binding a purification handle.
95 . A method of preventing or treating a disease in a subject in need thereof comprising introducing an effective amount of the RNA molecule of claim 70 .
96 . A compound of Formula (III) or (IV):
wherein N is a nucleoside;
L is a linker capable of binding a purification handle;
P is a purification handle;
Q-L1 is optionally present, wherein L1 is a linker covalently bound to N and to Q; and
Q is a hydrogen or a chain terminating nucleoside.
97 . The compound of claim 96 , wherein the nucleoside is a modified nucleoside.
98 . The compound of claim 96 , wherein the purification handle comprises a lipid.
99 . A method of increasing the expression of a protein or a peptide of interest in a cell, or of increasing the half-life of an RNA molecule in a cell, comprising contacting the cell with an RNA molecule comprising the compound of Formula (III) or Formula (IV) of claim 97 , wherein the RNA molecule encodes the protein or peptide of interest, wherein the expression is increased when compared to that of an RNA molecule without the compound of Formula (III) or Formula (IV), optionally wherein the cell is isolated, in vitro, or ex vivo, or the half-life is increased when compared to that of an RNA molecule without the compound of Formula (III) or Formula (IV), optionally wherein the cell is isolated, in vitro, or ex vivo.