Use of Thermostable RNA Polymerases to Produce RNAs Having Reduced Immunogenicity

Abstract

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.

What is claimed is: 
     
         1 . A composition comprising:
 (a) an RNA polymerase, wherein the RNA polymerase:
 (i) has at least 90% sequence identity to SEQ ID NO:1; and 
 (ii) having (1) at least 90% sequence identity to SEQ ID NO:1, and (2) at least one amino acid substitution corresponding to a position selected from the group consisting of: 109, 205, 534, 567, and 618 of SEQ ID NO:1; and 
   (b) a single-stranded RNA product having reduced immunogenicity compared with an RNA product produced by an RNA polymerase at 37° C.   
     
     
         2 . A composition according to  claim 1 , further comprising a pharmaceutically acceptable excipient. 
     
     
         3 . A composition according to  claim 1 , wherein the RNA polymerase has a further amino acid substitution at a position selected from positions 109, 205, 534, and 619. 
     
     
         4 . A composition according to  claim 1 , wherein the RNA polymerase has an amino acid substitution at a position corresponding to position 567, wherein the substation is V567P. 
     
     
         5 . A composition according to  claim 1 , wherein the RNA polymerase has an amino acid substitution at a position corresponding to position 388. 
     
     
         6 . A composition according to  claim 1 , wherein the RNA product encodes a therapeutic protein. 
     
     
         7 . A composition according to  claim 1 , wherein the RNA product is a therapeutic RNA. 
     
     
         8 . A composition according to  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 . A composition according to  claim 1 , wherein the RNA product comprises one or more modified nucleotides. 
     
     
         10 . 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 at least one amino acid substitution corresponding to a position selected from the group consisting of: 109, 205, 534, and 618 of SEQ ID NO:1; 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.   
     
     
         11 . A method according to  claim 10 , further comprising combining the single stranded RNA with a pharmaceutically acceptable excipient. 
     
     
         12 . A method according to  claim 11 , further comprising introducing the formulation into a mammalian cell. 
     
     
         13 . The method of  claim 11 , further comprising administering the formulation to a subject. 
     
     
         14 . The method of  claim 10 , wherein the transcribing is done at a temperature of at least 50° C. 
     
     
         15 . The method of  claim 10 , wherein the RNA polymerase has a further amino acid substitution at a position selected from positions 109, 205, 534, and 619. 
     
     
         16 . The method of  claim 10 , wherein the RNA product encodes a therapeutic protein. 
     
     
         17 . The method of  claim 10 , wherein the RNA product is a therapeutic RNA. 
     
     
         18 . The method of  claim 10 , 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.