Targeted rna editing by leveraging endogenous adar using engineered rnas

Abstract

The present application provides methods for editing RNA by introducing a deaminase-recruiting RNA in a host cell for deamination of an adenosine in a target RNA. The present application further provides deaminase-recruiting RNAs used in the RNA editing methods and compositions and kits comprising the same.

1 - 43 . (canceled) 
     
     
         44 . A method of modulating splicing in a target pre-messenger RNA comprising a target adenosine in a host cell, comprising introducing a deaminase-recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding the dRNA into the host cell, wherein:
 (1) the dRNA comprises a targeting RNA sequence that is at least partially complementary to the target pre-messenger RNA;   (2) the dRNA is a circular RNA or capable of forming a circular RNA within the host cell; and   (3) the dRNA hybridizes with the target pre-messenger RNA and recruits ADAR to the target pre-messenger RNA, thereby modulating the splicing of the target pre-messenger RNA.   
     
     
         45 . The method of  claim 44 , wherein the method comprises introducing a construct comprising the nucleic acid encoding the dRNA into the host cell. 
     
     
         46 . The method of  claim 45 , wherein the dRNA forms a circular RNA within the host cell. 
     
     
         47 . The method of  claim 46 , wherein the dRNA further comprises a 3′ ligation sequence and a 5′ ligation sequence. 
     
     
         48 . The method of  claim 46 , wherein the dRNA is circularized by RNA ligase RtcB in the host cell. 
     
     
         49 . The method of  claim 45 , wherein the construct further comprises a 3′ ribozyme sequence linked to the 3′ end of the nucleic acid encoding the dRNA and a 5′ ribozyme sequence linked to the 5′ end of the nucleic acid encoding the dRNA. 
     
     
         50 . The method of  claim 49 , wherein the 3′ ribozyme sequence is a 3′ twister ribozyme sequence and the 5′ ribozyme sequence is a 5′ twister ribozyme sequence. 
     
     
         51 . The method of  claim 50 , wherein:
 (i) the 3′ twister ribozyme sequence is twister P3 U2A and the 5′ twister ribozyme sequence is twister P1; or   (ii) the 5′ twister ribozyme sequence is twister P3 U2A and the 3′ twister ribozyme sequence is twister P1.   
     
     
         52 . The method of  claim 45 , wherein the construct further comprises a promoter operably linked to the nucleic acid encoding the dRNA, wherein the promoter is a polymerase II promoter (“Pol II promoter”) or a polymerase III promoter (“Pol III promoter”). 
     
     
         53 . The method of  claim 52 , wherein the Pol II promoter is a CMV promoter. 
     
     
         54 . The method of  claim 52 , wherein the Pol III promoter is a U6 promoter. 
     
     
         55 . The method of  claim 45 , wherein the construct is an adeno-associated virus (AAV) vector. 
     
     
         56 . The method of  claim 44 , wherein the ADAR is endogenously expressed by the host cell. 
     
     
         57 . The method of  claim 44 , wherein:
 (i) the targeting RNA sequence is more than 50 nucleotides in length;   (ii) the targeting RNA sequence comprises a cytidine, adenosine, or uridine directly opposite the target adenosine in the target pre-messenger RNA;   (iii) the targeting RNA sequence further comprises one or more guanosines each opposite a non-target adenosine in the target pre-messenger RNA;   (iv) the targeting RNA sequence comprises two or more consecutive mismatch nucleotides opposite a non-target adenosine in the target pre-messenger RNA;   (v) the 5′ nearest neighbor of the target adenosine in the target pre-messenger RNA is a nucleotide selected from U, C, A, and G with the preference of U>C≈A>G, and the 3′ nearest neighbor of the target adenosine in the target pre-messenger RNA is a nucleotide selected from G, C, A, and U with the preference of G>C>A≈U; and/or   (vi) the target adenosine is in a three-base motif selected from the group consisting of UAG, UAC, UAA, UAU, CAG, CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC, GAA, and GAU in the target pre-messenger RNA.   
     
     
         58 . The method of  claim 44 , comprising introducing a plurality of dRNAs each targeting a different target pre-messenger RNA or constructs comprising nucleic acids encoding the dRNAs each targeting a different target pre-messenger RNA. 
     
     
         59 . The method of  claim 44 , wherein the host cell is a mammalian cell. 
     
     
         60 . The method of  claim 44 , wherein deamination of the target adenosine in the target pre-messenger RNA by the recruited ADAR results in generation of an aberrant splice site and/or alternative splice site, or reversal of an aberrant splice site and/or alternative splice site. 
     
     
         61 . The method of  claim 44 , comprising introducing a dRNA into the host cell. 
     
     
         62 . The method of  claim 61 , wherein the dRNA is a circular RNA. 
     
     
         63 . A method for treating a disease or condition associated with a target pre-messenger RNA in an individual, comprising editing the target pre-messenger RNA in a cell of the individual according to the method of  claim 44 .