Use of thermostable RNA polymerases to produce RNAs having reduced immunogenicity
Provided herein, among other things, is a method for producing an RNA product that has reduced immunogenicity. In some embodiments, the method involves transcribing a template DNA with a thermostable RNA polymerase at a temperature of greater than 44° C.
1. A method for producing an RNA product with reduced immunogenicity, comprising:
(a) combining a DNA and a thermostable RNA polymerase at a temperature of greater than 44° C., the RNA polymerase variant having an amino acid sequence that has at least 90% sequence identity with SEQ ID NO:1 and having an amino acid substitution at a position corresponding to position 567 and optionally at a position corresponding to position 388; and
(b) producing the RNA product comprising a single strand RNA having reduced immunogenicity compared with an RNA product produced by an RNA polymerase at 37° C.
2. A method according to claim 1 , further comprising combining the single stranded RNA with a pharmaceutically acceptable excipient.
3. A method according to claim 2 , further comprising introducing the formulation into a mammalian cell.
4. The method of claim 2 , further comprising administering the formulation to a subject.
5. The method of claim 1 , wherein the transcribing is done at a temperature of at least 50° C.
6. The method of claim 1 , wherein the thermostable RNA polymerase has a further amino acid substitution at a position selected from positions 109, 205, 534, and 619.
7. The method of claim 1 , wherein the thermostable RNA polymerase has an amino acid substitution at a position corresponding to position 567, wherein the substation is V567P.
8. The method of claim 1 , wherein the RNA product encodes a therapeutic protein.
9. The method of claim 1 , wherein the RNA product is a therapeutic RNA.
10. The method of claim 1 , wherein the RNA product is selected from a guide RNA, a short hairpin RNA, a siRNA, a microRNA, a long noncoding RNA, a mRNA encoding a recombinant protein or a native protein, an RNA containing modified nucleotides, and a capped mRNA.
11. The method of claim 1 further comprises producing the RNA product comprising a single strand RNA having reduced immunogenicity in the absence of a step that removes any double-stranded RNA (dsRNA) from the RNA product.