Purification and Purity Assessment of RNA Molecules Synthesized with Modified Nucleosides

Abstract

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.

What is claimed is: 
     
         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.