POLYRIBONUCLEOTIDES CONTAINING REDUCED URACIL CONTENT AND USES THEREOF
The invention related to polyribonucleotides comprising an open reading frame of linked nucleosides encoding a polypeptide of interest (e.g., a therapeutic polypeptide), isoforms thereof, functional fragments thereof, and fusion proteins comprising the polypeptide. In some embodiments, the open reading frame is sequence-optimized. In particular embodiments, the invention provides sequence-optimized polyribonucleotides comprising nucleotides encoding the sequence of the polypeptide of interest, or sequence having high sequence identity with those sequence optimized polyribonucleotides.
1 . A polyribonucleotide comprising an open reading frame (ORF) encoding a therapeutic polypeptide, wherein the uracil content of the ORF relative to the theoretical minimum uracil content of a nucleotide sequence encoding the therapeutic polypeptide (% U TM ), is between about 100%/o and about 150%; and wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the uracils are 5-methoxyuracils.
2 . The polyribonucleotide of claim 1 , wherein the % U TM is between about 105% and about 145%, between about 105% and about 140%, between about 110% and about 140%, between about 110% and about 145%, between about 115% and about 135%, between about 105% and about 135%, between about 110% and about 135%, between about 115% and about 145%, or between about 115% and about 140%.
3 . (canceled)
4 . The polyribonucleotide of claim 1 , wherein the uracil content of the ORF relative to the uracil content of the corresponding wild-type ORF (% U WT ) is less than 100%.
5 . The polyribonucleotide of claim 4 , wherein the % U WT is less than about 95%, less than about 90%, less than about 85%, less than 80%, less than 79%, less than 78%, less than 77%, less than 76%, less than 75%, less than 74%, or less than 73%.
6 . (canceled)
7 . The polyribonucleotide of claim 1 , wherein the uracil content in the ORF relative to the total nucleotide content in the ORF (% U TL ) is less than about 50%, less than about 40%, less than about 30%, or less than about 19%.
8 - 9 . (canceled)
10 . The polyribonucleotide of claim 1 , wherein the guanine content of the ORF with respect to the theoretical maximum guanine content of a nucleotide sequence encoding the polypeptide (% G TMX ) is at least 69%, at least 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
11 . (canceled)
12 . The polyribonucleotide of claim 1 , wherein the cytosine content of the ORF relative to the theoretical maximum cytosine content of a nucleotide sequence encoding the polypeptide (% C TMX ) is at least 59%, at least 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
13 . (canceled)
14 . The polyribonucleotide of claim 1 , wherein the guanine and cytosine content (G/C) of the ORF relative to the theoretical maximum G/C content in a nucleotide sequence encoding the polypeptide (% G/C TMX ) is at least about 81%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
15 . (canceled)
16 . The polyribonucleotide of claim 1 , wherein the G/C content in the ORF relative to the G/C content in the corresponding wild-type ORF (% G/C WT ) is at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 110%, at least 115%, or at least 120%.
17 . The polyribonucleotide of claim 1 , wherein the average G/C content in the 3 rd codon position in the ORF is at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, or at least 30% higher than the average G/C content in the 3 rd codon position in the corresponding wild-type ORF.
18 . The polyribonucleotide of claim 1 , wherein the ORF further comprises at least one low-frequency codon.
19 . The polyribonucleotide of claim 1 , wherein the polyribonucleotide sequence further comprises a nucleotide sequence encoding a transit peptide.
20 . (canceled)
21 . The polyribonucleotide of claim 1 , wherein the polyribonucleotide further comprises at least one chemically modified nucleobase, sugar backbone, or any combination thereof, in addition to 5-methoxyuridine.
22 . The polyribonucleotide of claim 21 , wherein the at least one chemically modified nucleobase is selected from the group consisting of pseudouracil (ψ), N1-methylpseudouracil (m1ψ), N1-ethyipseudouracil (Et1ψ), 2-thiouracil (s2U), 4′-thiouracil, 5-methylcytosine, 5-methyluracil, and any combination thereof.
23 . The polyribonucleotide of claim 1 , wherein the polyribonucleotide further comprises a miRNA binding site.
24 - 37 . (canceled)
38 . The polyribonucleotide of claim 1 , wherein the polyribonucleotide encodes a therapeutic polypeptide that is fused to one or more heterologous polypeptides.
39 - 40 . (canceled)
41 . The polyribonucleotide of claim 1 , wherein the polyribonucleotide comprises:
(i) a 5′-terminal cap;
(ii) a 5′-UTR;
(iii) an ORF encoding a therapeutic polypeptide;
(iv) a 3′-UTR; and
(v) a poly-A region.
42 . (canceled)
43 . A method of producing the polyribonucleotide of claim 1 , the method comprising modifying an ORF encoding a therapeutic polypeptide by substituting at least one uracil nucleobase with an adenine, guanine, or cytosine nucleobase, or by substituting at least one adenine, guanine, or cytosine nucleobase with a uracil nucleobase, wherein all the substitutions are synonymous substitutions.
44 . A composition comprising
(a) the polyribonucleotide of claim 1 ; and
(b) a delivery agent.
45 - 76 . (canceled)
77 . A host cell comprising the polyribonucleotide of claim 1 .
78 . (canceled)
79 . A vector comprising the polyribonucleotide of claim 1 .
80 . A method of making a polyribonucleotide comprising enzymatically or chemically synthesizing the polyribonucleotide of claim 1 .
81 . A method of expressing in vivo an active therapeutic polypeptide in a subject in need thereof comprising administering to the subject an effective amount of the polyribonucleotide of claim 1 .
82 - 84 . (canceled)
85 . The method of claim 81 , wherein the polyribonucleotide does not lead to an increase in B-cell frequency when administered to the subject, compared to B-cell frequencies measured in the subject in the absence of the polyribonucleotide.
86 . (canceled)
87 . The method of claim 81 , wherein the polyribonucleotide does not lead to an activation of CD86 when administered to the subject.
88 - 111 . (canceled)
112 . The polyribonucleotide of claim 1 , wherein the polyribonucleotide comprises at least two different microRNA (miR) binding sites, wherein the microRNA is expressed in an immune cell of hematopoietic lineage or a cell that expresses TLR7 and/or TLR8 and secretes pro-inflammatory cytokines and/or chemokines, and wherein the polyribonucleotide comprises one or more modified nucleobases.
113 - 118 . (canceled)
119 . The polyribonucleotide of claim 112 , wherein the polyribonucleotide comprises at least one first microRNA binding site of a microRNA abundant in an immune cell of hematopoietic lineage and at least one second microRNA binding site is of a microRNA abundant in endothelial cells.
120 - 163 . (canceled)