Large-scale synthesis of messenger rna

Abstract

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.