Manufacturing methods for production of rna transcripts

Abstract

Described are methods for production of RNA transcripts using a non-amplified, linearized DNA tempate in an in vitro transcription reaction. Enzymatic 5′ capping and oligo dT purification can also be included in the methods.

1 . A method for producing a purified composition comprising a capped RNA transcript for a gene of interest, the method comprising:
 (a) providing a sample comprising a linear, non-amplified DNA template, the DNA template comprising an RNA polymerase promoter sequence operatively linked to a target sequence coding for the gene of interest, a poly A tail sequence, an endonuclease recognition site sequence immediately downstream of the poly A tail sequence, and optionally a 5′ untranslated region (UTR) and/or a 3′ UTR;   (b) contacting the sample with a RNA polymerase and ribonucleotides   under conditions sufficient for vitro transcription to produce a first composition comprising an uncapped RNA transcript, wherein at least 80% of the RNA transcript is full-length uncapped RNA transcript;   (c) purifying the uncapped RNA transcript using oligo dT affinity purification;   (d) capping the uncapped RNA transcript to produce a second composition comprising a capped RNA transcript; and   (e) purifying the capped RNA transcript from the second composition by anion exchange chromatography,   thereby producing the purified composition comprising a capped RNA transcript.   
     
     
         2 . The method of  claim 1 , wherein at least 95% of the uncapped RNA transcript in the first composition is full-length uncapped RNA transcript. 
     
     
         3 . The method of  claim 1 , wherein the percent full length uncapped RNA transcript is determined using reverse phase HPLC and measured by peak area of full length relative to total peak area. 
     
     
         4 . The method of  claim 1 , wherein the method does not comprising treating the composition with DNase. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein the method does not comprise a polymerase chain reaction (PCR) amplification step. 
     
     
         7 . The method of  claim 1 , wherein the DNA template comprises 500-25000 or 3000-7000 basepairs. 
     
     
         8 . (canceled) 
     
     
         9 . The method of  claim 1 , wherein the poly A tail is 5-300 nucleotides or 60-160 nucleotides in length 
     
     
         10 . The method of  claim 1 , wherein the endonuclease recognition site sequence is recognized by XbaI or SapI. 
     
     
         11 . The method of  claim 1 , wherein the method further comprises producing the DNA template, wherein producing the DNA template comprises contacting a circular plasmid DNA template with an endonuclease that recognizes the endonuclease recognition site sequence. 
     
     
         12 . The method of  claim 11 , wherein the method further comprises producing the circular plasmid DNA template at a microgram scale or a milligram scale or a gram scale. 
     
     
         13 . The method of  claim 1 , wherein the RNA polymerase is a T7 polymerase. 
     
     
         14 . The method of  claim 1 , wherein at least one nucleotide is a modified nucleotide. 
     
     
         15 . The method of  claim 1 , wherein capping the uncapped RNA transcript comprises contacting the RNA transcript with Vaccinia guanylyltransferase, s-adenosyl-L-methionine (SAM), and guanosine triphosphate (GTP) in a reaction and maintaining the reaction under conditions sufficient to cap the RNA transcript, wherein the RNA transcript is capped with a Cap0 structure. 
     
     
         16 . The method of  claim 15 , wherein the reaction further comprises 2′-O-methyltransferase wherein the RNA transcript is capped with a Cap1 structure. 
     
     
         17 - 22 . (canceled) 
     
     
         23 . The method of  claim 1 , wherein the DNA template comprises a poly A tail sequence of 60-160 nucleotides; the RNA polymerase is a T7 RNA polymerase; and capping the uncapped RNA transcript comprises contacting the uncapped RNA transcript with guanylyltransferase, s-adenosyl-L-methionine (SAM), guanosine triphosphate (GTP), and optionally 2′-O-methyltransferase.