Purification and Purity Assessment of RNA Molecules Synthesized with Modified Nucleosides
This invention provides purified preparations of an RNA, oligoribonucleotide, or polyribonucleotide comprising a modified nucleoside, and methods of assessing purity of purified preparations of an RNA, oligoribonucleotide, or polyribonucleotide comprising a modified nucleoside.
1 . A purified preparation of messenger RNA comprising a 1-methyl-pseudouridine residue.
2 . The purified preparation of messenger RNA of claim 1 , further comprising a poly-A tail.
3 . The purified preparation of messenger RNA of claim 1 , further comprising an m7GpppG cap.
4 . The purified preparation of messenger RNA of claim 1 , further comprising a cap-independent translational enhancer.
5 . The purified preparation of messenger RNA of claim 1 , wherein the messenger RNA comprises at least about 95% to about 99.9% of all the nucleic acid present in the purified preparation.
6 . The purified preparation of messenger RNA of claim 1 , wherein the messenger RNA is significantly less immunogenic than an unpurified preparation of messenger RNA with the same sequence.
7 . The purified preparation of messenger RNA of claim 1 , wherein the messenger RNA exhibits enhanced ability to be translated by a target cell than an unpurified preparation of messenger RNA with the same sequence.
8 . The purified preparation of messenger RNA of claim 1 , wherein the messenger RNA exhibits enhanced ability to be translated when delivered to a mammal than an unpurified preparation of messenger RNA with the same sequence.
9 . The purified preparation of messenger RNA of claim 1 , wherein the messenger RNA is encapsulated in a lipid nanoparticle.
10 . The purified preparation of messenger RNA of claim 1 , wherein the modified nucleoside is at least one of 1-methyl-pseudouridine and m5C.
11 . The purified preparation of messenger RNA of claim 1 , wherein the modified nucleoside is at least one of pseudouridine and m5C.
12 . A purified preparation of RNA encoding a protein of interest, the RNA comprising at least one of a 1-methyl-pseudouridine residue and a m5C residue.
13 . A purified preparation of RNA encoding a protein of interest, the RNA comprising at least one of a pseudouridine residue and a m5C residue.
14 . The purified preparation of RNA of claim 13 , further comprising a poly-A tail.
15 . The purified preparation of RNA of claim 13 , further comprising an m7GpppG cap.
16 . The purified preparation of RNA of claim 13 , further comprising a cap-independent translational enhancer.
17 . The purified preparation of RNA of claim 13 , wherein the messenger RNA comprises at least about 95% to about 99.9% of all the nucleic acid present in the purified preparation.
18 . The purified preparation of RNA of claim 13 , whereby the messenger RNA is significantly less immunogenic than an unpurified preparation of messenger RNA with the same sequence.
19 . The purified preparation of RNA of claim 13 , wherein the RNA exhibits enhanced ability to be translated by a target cell than an unpurified preparation of messenger RNA with the same sequence.
20 . The purified preparation of RNA of claim 13 , wherein the RNA exhibits enhanced ability to be translated when delivered to a mammal than an unpurified preparation of messenger RNA with the same sequence.
21 . The purified preparation of RNA of claim 13 , wherein the RNA is encapsulated in a lipid nanoparticle.
22 . The purified preparation of RNA of claim 13 , wherein the protein of interest is VEGF-A.
23 . The purified preparation of RNA of claim 13 , wherein the protein of interest is erythropoietin (EPO).
24 . A method of preparing a purified preparation of messenger RNA comprising at least one modified nucleoside comprising the steps of:
a. producing a preparation of messenger RNA comprising at least one modified nucleoside;
b. subjecting the preparation of messenger RNA comprising at least one modified nucleoside to at least one purification process selected from the group consisting of enzyme digestion and chromatography; and
c. isolating the purified preparation of messenger RNA comprising at least one modified nucleoside.
25 . The method of claim 24 , wherein the at least one modified nucleoside is 1-methyl-pseudouridine.
26 . The method of claim 24 , wherein the messenger RNA further comprises a poly-A tail.
27 . The method of claim 24 , wherein the messenger RNA further comprises an m7GpppG cap.
28 . The method of claim 24 , wherein the messenger RNA further comprises a cap-independent translational enhancer.
29 . The method of claim 24 , wherein the messenger RNA comprises at least about 95% to about 99.9% of all the nucleic acid present in the purified preparation.
30 . The method of claim 24 , wherein the messenger RNA is significantly less immunogenic than an unpurified preparation of messenger RNA with the same sequence.
31 . The method of claim 24 , wherein the messenger RNA exhibits enhanced ability to be translated by a target cell than an unpurified preparation of messenger RNA with the same sequence.
32 . The method of claim 24 , wherein the messenger RNA exhibits enhanced ability to be translated when delivered to a mammal than an unpurified preparation of messenger RNA with the same sequence.
33 . The method of claim 24 , wherein the modified nucleoside comprises at least one of 1-methyl-pseudouridine and m5C.
34 . The method of claim 24 , wherein the modified nucleoside comprises at least one of pseudouridine and m5C.
35 . The method of claim 24 , wherein the preparation of messenger RNA is produced by in vitro transcription.
36 . The method of claim 24 , wherein the at least one purification process is enzyme digestion.
37 . The method of claim 36 , wherein the enzyme digestion is performed using at least one enzyme selected from the group consisting of RNase III, RNase V1, Dicer, and Chipper.
38 . The method of claim 24 , wherein the at least one purification process is chromatography.
39 . The method of claim 38 , wherein the chromatography is at least one selected from the group consisting of high performance liquid chromatography (HPLC) and fast protein liquid chromatography (FPLC).
40 . A method for inducing a mammalian cell to produce a protein of interest, the method comprising the step of contacting the mammalian cell with the purified preparation of the RNA of claim 1 , thereby inducing a mammalian cell to produce a protein of interest.
41 . The method of claim 40 , wherein the mammalian cell is a dendritic cell.
42 . The method of claim 40 , wherein the mammalian cell is an alveolar cell, an astrocyte, a microglial cell, or a neuron.
43 . A purified preparation of in vitro-transcribed RNA, comprising a at least one modified nucleoside.
44 . The purified preparation of in vitro-transcribed RNA of claim 43 , wherein the at least one modified nucleoside is 1-methyl-pseudouridine, m5C, m5U, m6A, s2U, Ψ, or 2′-O-methyl-U.
45 . The purified preparation of in vitro-transcribed RNA of claim 43 , further comprising a poly-A tail.
46 . The purified preparation of in vitro-transcribed RNA of claim 43 , further comprising an m7GpppG cap.
47 . The purified preparation of in vitro-transcribed RNA of claim 43 , further comprising a cap-independent translational enhancer.
48 . A purified preparation of an in vitro-synthesized oligoribonucleotide, comprising at least one modified nucleoside, wherein the at least one modified nucleoside is 1-methyl-pseudouridine, m5C, m5U, m6A, s2U, Ψ, or 2′-O-methyl-U.
49 . The purified preparation of in vitro-synthesized oligoribonucleotide of claim 48 , wherein the in vitro-synthesized oligoribonucleotide is a therapeutic oligoribonucleotide.
50 . A purified preparation of a gene-therapy vector, comprising an in vitro-synthesized polyribonucleotide encoding a protein of interest, wherein the polyribonucleotide comprises at least one modified nucleoside.
51 . The purified preparation of gene-therapy vector of claim 50 , wherein the at least one modified nucleoside is 1-methyl-pseudouridine, m5C, m5U, m6A, s2U, Ψ, or 2′-O-methyl-U.
52 . The purified preparation of gene-therapy vector of claim 50 , wherein the polyribonucleotide further comprises a poly-A tail.
53 . The purified preparation of gene-therapy vector of claim 50 , wherein the polyribonucleotide further comprises an m7GpppG cap.
54 . The purified preparation of gene-therapy vector of claim 50 , wherein the polyribonucleotide further comprises a cap-independent translational enhancer.
55 . A method for delivering a recombinant protein to a subject, the method comprising the step of contacting a cell of the subject with the purified preparation of the gene-therapy vector of claim 50 , wherein the cell produces the recombinant protein, thereby delivering a recombinant protein to a subject.
56 . The method of claim 55 , wherein the cell is a dendritic cell.
57 . The method of claim 55 , wherein the cell is a lung cell, a brain cell, or a spleen cell.