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 messenger RNA that comprises an open reading frame, the method comprising:
(a) forming a reaction mixture comprising a DNA template and nucleoside triphosphates (NTPs) including adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), guanosine triphosphate (GTP), or an analog of each respective nucleoside, and a buffer; and
(b) incubating the reaction mixture to produce a composition comprising messenger RNA,
wherein the ratio of concentration of GTP to concentration of ATP is at least 2:1, the ratio of concentration of GTP to concentration of CTP is at least 2:1, and the ratio of concentration of GTP to concentration of UTP is at least 2:1.
2. The method of claim 1 , wherein the reaction mixture comprises GTP and GDP and wherein the ratio of concentration of GTP plus GDP to the concentration of any one of ATP, CTP or UTP is at least 2:1.
3. The method of claim 2 , wherein the ratio of concentration of GTP plus GDP to concentration of ATP, CTP and UTP is at least 3:1, at least 6:1, and at least 6:1, respectively.
4. The method of claim 1 , wherein the reaction mixture further comprises guanosine diphosphate (GDP).
5. The method of claim 1 , wherein the composition is substantially free of reverse complement transcription product.
6. The method of claim 1 , wherein greater than 90% of the mass of the messenger RNA comprises single stranded full length transcripts.
7. The method of claim 1 , wherein the composition is substantially free of RNase III insensitive fragments.
8. The method of claim 1 , wherein the composition comprises a population of single stranded partial RNA transcripts in a sense orientation and wherein greater than 80% of the population of single stranded partial RNA transcripts in a sense orientation has a nucleotide length of 100 nucleotides or shorter.
9. The method of claim 8 , wherein greater than 90% of the population of single stranded partial RNA transcripts in a sense orientation has a nucleotide length of 100 nucleotides or shorter.
10. The method of claim 1 , wherein less than 0.25% of the mass of the RNA in the composition is cytokine-inducing RNA contaminant.
11. The method of claim 10 , wherein the composition is substantially free of cytokine-inducing RNA contaminant.
12. The method of claim 1 , wherein less than 0.5% of the mass of the RNA in the composition is reverse complement transcription product.
13. The method of claim 12 , wherein the reverse complement transcription product is a dsRNA comprising a strand comprising a sequence that is a reverse complement of at least a portion of the messenger RNA or a dsRNA comprising a strand comprising a polyU containing sequence.
14. The method of claim 13 , wherein the strand comprising the sequence that is the reverse complement of the messenger RNA or the strand comprising the polyU sequence initiates with a 5′ triphosphate (5′-PPP).
15. The method of claim 12 , wherein the reverse complement transcription product comprises a reverse complement of the 5′-end of the messenger RNA and/or a reverse complement of the 3′-end of the messenger RNA encoding a polypeptide of interest.
16. The method of claim 1 , wherein the messenger RNA is an RNA encoding a polypeptide of interest and wherein the reverse complement of the messenger RNA encoding the polypeptide of interest comprises a sequence complementary to all or a portion of an open reading frame of the messenger RNA encoding the polypeptide of interest.
17. A method of producing messenger RNA that comprises an open reading frame, the method comprising:
(a) forming a reaction mixture comprising a DNA template and nucleoside triphosphates (NTPs) including adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), guanosine triphosphate (GTP), or an analog of each respective nucleoside, and a buffer; and
(b) incubating the reaction mixture to produce a composition comprising messenger RNA,
wherein the ratio of concentration of GTP to concentration of ATP is at least 2:1, the ratio of concentration of GTP to concentration of CTP is at least 4:1, and the ratio of concentration of GTP to concentration of UTP is at least 4:1.
18. The method of claim 17 , wherein the ratio of concentration of GTP to concentration of ATP is 2:1, the ratio of concentration of GTP to concentration of CTP is 4:1, and the ratio of concentration of GTP to concentration of UTP is 4:1.