High purity RNA compositions and methods for preparation thereof
The invention relates to improved RNA compositions for use in therapeutic applications. The RNA compositions are particularly suited for use in human therapeutic application (e.g., in RNA therapeutics). The RNA compositions are made by improved processes, in particular, improved in vitro-transcription (IVT) processes. The invention also relates to methods for producing and purifying RNA (e.g, therapeutic RNAs), as well as methods for using the RNA compositions and therapeutic applications thereof.
1. A method of producing a messenger ribonucleic acid (mRNA) that comprises an open reading frame encoding a vaccine antigen, the method comprising incubating a reaction mixture comprising a deoxyribonucleic acid (DNA), an RNA polymerase, nucleoside triphosphates (NTPs), and buffer,
wherein the NTPs comprise adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), and guanosine triphosphate (GTP), or an analog of each respective NTP, and
wherein the reaction mixture comprises the GTP or an analogue thereof in an amount greater than an amount of one or more of the other NTPs or analogues thereof,
thereby producing a composition comprising the mRNA.
2. The method of claim 1 , wherein the amount of the GTP or an analogue thereof is greater than an amount of the UTP or an analogue thereof.
3. The method of claim 1 , wherein the amount of the GTP is greater than the amount of the UTP.
4. The method of claim 3 , wherein an amount of the ATP is greater than the amount of the UTP, and the amount of the CTP is greater than the amount of the UTP.
5. The method of claim 1 , wherein the reaction mixture comprises an at least 2:1 ratio of GTP concentration to ATP concentration, an at least 2:1 ratio of GTP concentration to CTP concentration, and an at least 4:1 ratio of GTP concentration to UTP concentration.
6. The method of claim 1 , wherein the reaction mixture comprises a 2:1 ratio of GTP concentration to ATP concentration, a 2:1 ratio of GTP concentration to CTP concentration, and a 4:1 ratio of GTP concentration to UTP concentration.
7. The method of claim 1 , wherein the reaction mixture further comprises guanosine diphosphate (GDP).
8. The method of claim 1 , wherein the reaction mixture comprises an at least 3:1 ratio of GTP plus GDP concentration to ATP concentration, an at least 6:1 ratio of GTP plus GDP concentration to CTP concentration, and an at least 6:1 ratio of GTP plus GDP concentration to UTP concentration.
9. The method of claim 1 , wherein the RNA polymerase is selected from T7 polymerase, T3 polymerase, and SP6 polymerase.
10. The method of claim 9 , wherein the RNA polymerase is T7 polymerase.
11. The method of claim 1 , wherein the DNA is cDNA.
12. The method of claim 1 , wherein the buffer comprises magnesium.
13. The method of claim 12 , wherein the reaction mixture further comprises dithiothreitol (DTT).
14. The method of claim 13 , wherein the reaction mixture further comprises spermidine.
15. The method of claim 14 , wherein the reaction mixture further comprises an RNase inhibitor.
16. The method of claim 14 , wherein the reaction mixture further comprises pyrophosphatase.
17. The method of claim 1 , wherein the reaction mixture is incubated at 25 degrees Celsius or at 37 degrees Celsius.
18. The method of claim 1 , wherein the produced mRNA is less immunogenic than an mRNA made by a comparable method but with equimolar amounts of the NTPs in the reaction mixture.
19. The method of claim 1 , wherein the produced mRNA has less double stranded impurities than an mRNA made by a comparable method but with equimolar amounts of the NTPs in the reaction mixture.
20. The method of claim 1 , wherein the reaction mixture does not contain an RNase inhibitor.
21. The method of claim 1 , wherein less than 1% of the total mass of RNA in the composition is a reverse complement transcription product.
22. The method of claim 21 , wherein less than 0.1% of the total mass of RNA in the composition is a reverse complement transcription product.
23. A method of producing a messenger ribonucleic acid (mRNA) that comprises an open reading frame encoding a therapeutic protein or peptide, the method comprising incubating a reaction mixture comprising a deoxyribonucleic acid (DNA), an RNA polymerase, nucleoside triphosphates (NTPs), and buffer,
wherein the NTPs comprise adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), and guanosine triphosphate (GTP), or an analog of each respective NTP, and
wherein the reaction mixture comprises the GTP or an analogue thereof in an amount greater than an amount of one or more of the other NTPs or analogues thereof,
thereby producing a composition comprising the mRNA.