LARGE-SCALE SYNTHESIS OF MESSENGER RNA
The present invention provides methods for large-scale production of a composition enriched for full-length mRNA molecules using an SP6 RNA polymerase and compositions produced using such methods and uses thereof.
1 . A method for large-scale production of a composition enriched for full-length mRNA molecules comprising synthesizing in vitro mRNA using an SP6 RNA polymerase, wherein at least 80% of the synthesized mRNA molecules are full-length, and wherein at least 100 mg of mRNA is synthesized in a single batch.
2 - 4 . (canceled)
5 . The method of claim 1 , wherein the composition is substantially free of abortive transcripts.
6 . The method of claim 5 , wherein the full-length or abortive transcripts of mRNA are detected by agarose gel electrophoresis or capillary electrophoresis.
7 . (canceled)
8 . The method of claim 6 , wherein the synthesized mRNA contains undetectable amount of abortive transcripts on Glyoxal agarose gel electrophoresis.
9 - 11 . (canceled)
12 . The method of claim 6 , wherein the relative amount of full-length or abortive transcripts of synthesized mRNA is determined by the relative peak areas corresponding to the full-length or abortive transcripts.
13 . The method of claim 12 any one of the preceding claims, wherein the full-length or abortive transcripts of mRNA are detected prior to capping or tailing the synthesized mRNA.
14 - 15 . (canceled)
16 . The method of claim 1 , wherein the full-length mRNA molecule is at least 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 600 bases, 700 bases, 800 bases, 900 bases, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb in length.
17 . The method of claim 1 , wherein at least 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 5 g, 10 g, 25 g, 50 g, 75 g, 100 g, 150 g, 200 g, 250 g, 500 g, 750 g, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg, 1000 kg, or more of mRNA is synthesized in a single batch.
18 - 20 . (canceled)
21 . The method of claim 1 , wherein the SP6 RNA polymerase is a recombinant SP6 RNA polymerase.
22 . The method of claim 21 , wherein the SP6 RNA polymerase comprises a tag.
23 . (canceled)
24 . The method of claim 1 , wherein the mRNA is synthesized by the SP6 RNA polymerase based on a DNA template.
25 . The method of claim 24 , wherein the DNA template comprises an SP6 promoter operably linked to a DNA sequence encoding the mRNA sequence to be synthesized.
26 . The method of claim 25 , wherein the DNA sequence is optimized.
27 . The method of claim 26 , wherein the DNA sequence is optimized to reduce the chance of a hairpin structure forming in the synthesized mRNA.
28 . The method of claim 1 , wherein the mRNA is synthesized in a reaction mixture comprising NTPs at a concentration ranging from 1-10 mM each NTP, the DNA template at a concentration ranging from 0.01-0.5 mg/ml, and the SP6 RNA polymerase at a concentration ranging from 0.01-0.1 mg/ml.
29 - 30 . (canceled)
31 . The method of any one of claim 28 , wherein the NTPs are naturally-occurring NTPs.
32 . The method of any one of claim 28 , wherein the NTPs comprise modified NTPs.
33 - 36 . (canceled)
37 . The method of claim 1 , wherein the mRNA is codon optimized.
38 . A composition comprising mRNA synthesized in vitro using an SP6 RNA polymerase, wherein at least 80% of the synthesized mRNA molecules are full-length, and wherein at least 100 mg of mRNA is synthesized in a single batch.
39 - 42 . (canceled)
43 . A method of delivering mRNA for in vivo protein production comprising administering a composition comprising mRNA synthesized in vitro using an SP6 RNA polymerase, wherein at least 80% of the synthesized mRNA molecules are full-length, and wherein at least 100 mg of mRNA is synthesized in a single batch.