Use of thermostable RNA polymerases to produce RNAs having reduced immunogenicity
View Patent ↗Provided herein, among other things, is a method for producing an RNA product that has reduced immunogenicity without requiring removal of any dsRNA from the RNA product. 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 comprising:
combining an RNA product made by transcribing a template DNA with a thermostable RNA polymerase at a temperature of greater than 44° C. with a pharmaceutically acceptable excipient, in the absence of a step that removes any dsRNA from the RNA product, to produce a formulation, and
introducing the formulation into a mammalian cell,
wherein the RNA product in the formulation is less immunostimulatory than a control RNA product produced by transcribing the template DNA with the polymerase at a temperature of 37° C.
2. The method of claim 1 , wherein the method comprises administering the formulation to a subject.
3. The method of claim 1 , wherein the transcribing is done at a temperature of at least 50° C.
4. The method of claim 1 , wherein the thermostable RNA polymerase is a variant of a bacteriophage RNA polymerase.
5. The method of claim 1 , wherein the thermostable RNA polymerase is a variant of the wild type T7 RNA polymerase of SEQ ID NO: 1.
6. The method of claim 1 , wherein the RNA product encodes a therapeutic protein.
7. The method of claim 1 , wherein the RNA product is a therapeutic RNA.
8. 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.
9. The method of claim 1 , wherein the mammalian cell is in vitro.
10. The method of claim 1 , wherein the mammalian cell is in vivo.
11. The method of claim 1 , wherein the mammalian cell is ex vivo.