Manufacturing methods for production of RNA transcripts
Described are methods for production of RNA transcripts using a non-amplified, linearized DNA template 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 of 60-160 nucleotides, an endonuclease recognition site sequence immediately downstream of the poly A tail sequence, and 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 by contacting the RNA transcript with quanlyltransferase, s-adenosyl-L-methionine, guanosine triphosphate, and 2′-O-methyltransferase to produce a second composition comprising a capped RNA transcript;
(e) purifying the capped RNA transcript from the second composition by anion exchange chromatography; and
(f) filtering the capped RNA transcript by ultrafiltration or tangential flow filtration, thereby producing the purified composition comprising a capped RNA transcript,
wherein the method does not comprise treating the composition with DNase.
2. The method of claim 1 , wherein at least 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 by 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 comprise a polymerase chain reaction (PCR) amplification step.
5. The method of claim 1 , wherein the endonuclease recognition site sequence is recognized by Xbal or Sapl.
6. 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.
7. The method of claim 6 , wherein the method further comprises producing the circular plasmid DNA template at a microgram scale, a milligram scale, or a gram scale.
8. The method of claim 1 , wherein the RNA polymerase is a T7polymerase.
9. The method of claim 1 , wherein at least one nucleotide is a modified nucleotide.