Targeted rna editing by leveraging endogenous adar using engineered rnas

Abstract

Provided are methods for editing RNA by introducing a deaminase-recruiting RNA in a host cell for deamination of an adenosine in a target RNA, deaminase-recruiting RNAs used in the RNA editing methods, compositions and kits comprising the same.

1 . A method for editing a target RNA 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 RNA,   (2) the dRNA is capable of recruiting an adenosine deaminase acting on RNA (ADAR), and   (3) the dRNA is a circular RNA or capable of forming a circular RNA.   
     
     
         2 - 7 . (canceled) 
     
     
         8 . A method for editing a target RNA 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 the dRNA comprises:
 (1) a targeting RNA sequence that is at least partially complementary to the target RNA and   (2) a small nucleolar RNA (snoRNA) sequence linked to the 3′ and/or 5′ ends of the targeting RNA sequence;   and wherein the dRNA is capable of recruiting an adenosine deaminase acting on RNA (ADAR).   
     
     
         9 . The method of  claim 8 , wherein
 the dRNA comprises a snoRNA sequence linked to the 5′ end of the targeting RNA sequence (“5′ snoRNA sequence”).   
     
     
         10 . The method of  claim 8 , wherein the method comprises introducing a construct comprising a nucleic acid encoding the dRNA into the host cell. 
     
     
         11 . The method of  claim 10 , 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”). 
     
     
         12 . A method for editing a target RNA in a host cell, comprising introducing a construct comprising a nucleic acid encoding a deaminase-recruiting RNA (dRNA) into the host cell, wherein:
 (1) the dRNA comprises a targeting RNA sequence that is at least partially complementary to the target RNA,   (2) the dRNA is capable of recruiting an adenosine deaminase acting on RNA (ADAR), and   (3) the construct comprises a polymerase II promoter (“Pol II promoter”) operably linked to the nucleic acid encoding the dRNA.   
     
     
         13 . The method of  claim 11 , wherein the Pol II promoter is a CMV promoter. 
     
     
         14 . The method of  claim 8 , wherein the construct is a viral vector or a plasmid, optionally wherein the construct is an AAV vector. 
     
     
         15 . The method of  claim 8 , wherein the ADAR is endogenously expressed by the host cell. 
     
     
         16 . The method of  claim 8 , 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 RNA;   (iii) the targeting RNA sequence further comprises one or more guanosines each opposite a non-target adenosine in the target RNA;   (iv) the targeting RNA sequence comprises two or more consecutive mismatch nucleotides opposite a non-target adenosine in the target RNA;   (v) the 5′ nearest neighbor of the target adenosine in the target RNA is a nucleotide selected from U, C, A and G with the preference U>C≈A>G and the 3′ nearest neighbor of the target adenosine in the target RNA is a nucleotide selected from G, C, A and U with the preference G>C>A≈U;   (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 RNA; and/or   (vii) the target RNA is an RNA selected from the group consisting of a pre-messenger RNA, a messenger RNA, a ribosomal RNA, a transfer RNA, a long non-coding RNA and a small RNA.   
     
     
         17 . (canceled) 
     
     
         18 . The method of  claim 8 , comprising introducing a plurality of dRNAs or constructs each targeting a different target RNA. 
     
     
         19 . (canceled) 
     
     
         20 . The method of  claim 8 , wherein the construct or the dRNA does not induce immune response. 
     
     
         21 . The method of  claim 8 , further comprising introducing an ADAR to the host cell. 
     
     
         22 . The method of  claim 8 , wherein deamination of the target adenosine in the target RNA results in a missense mutation, an early stop codon, aberrant splicing, or alternative splicing in the target RNA, or reversal of a missense mutation, an early stop codon, aberrant splicing, or alternative splicing in the target RNA. 
     
     
         23 . The method of  claim 8 , wherein the host cell is a eukaryotic cell. 
     
     
         24 . An edited RNA or a host cell having an edited RNA produced by the method of  claim 8 . 
     
     
         25 . A method for treating or preventing a disease or condition in an individual, comprising editing a target RNA associated with the disease or condition in a cell of the individual according to the method of  claim 8 . 
     
     
         26 - 32 . (canceled) 
     
     
         33 . A construct, comprising a nucleic acid encoding a dRNA for editing a target RNA comprising a targeting RNA sequence that is at least partially complementary to the target RNA, wherein the dRNA is capable of recruiting an Adenosine Deaminase Acting on RNA (ADAR), and wherein the dRNA is circular or is capable of forming a circular RNA, and wherein the construct further comprises a 3′ twister ribozyme sequence linked to the 3′ end of the nucleic acid encoding the dRNA and a 5′ twister ribozyme sequence linked to the 5′ end of the nucleic acid encoding the dRNA, optionally wherein:
 (i) the 3′ twister sequence is twister P3 U2A and the 5′ twister sequence is twister P1; or 
 (ii) the 5′ twister sequence is twister P3 U2A and the 3′ twister sequence is twister P1. 
 
     
     
         34 . A deaminase-recruiting RNA (dRNA) for editing a target RNA comprising:
 (1) a targeting RNA sequence that is at least partially complementary to the target RNA and   (2) a small nucleolar RNA (snoRNA) sequence at the 3′ and/or 5′ ends of the targeting RNA sequence;   wherein the dRNA is capable of recruiting an adenosine deaminase acting on RNA (ADAR).   
     
     
         35 . (canceled) 
     
     
         36 . A construct comprising a nucleic acid encoding a dRNA of  claim 34 . 
     
     
         37 . The construct of  claim 36 , 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”). 
     
     
         38 - 41 . (canceled) 
     
     
         42 . A host cell comprising the dRNA  claim 34 . 
     
     
         43 . A kit for editing a target RNA in a host cell comprising the dRNA of  claim 34 . 
     
     
         44 . The method of  claim 8 , wherein the dRNA comprises a snoRNA sequence linked to the 3′ end of the targeting RNA sequence (3′ snoRNA sequence”). 
     
     
         45 . The method of  claim 8 , wherein the snoRNA sequence is at least about 70 nucleotides in length. 
     
     
         46 . The method of  claim 44 , wherein the 3′ snoRNA sequence comprises the nucleic acid sequence of SEQ ID NO: 1. 
     
     
         47 . The method of  claim 9 , wherein the 5′ snoRNA sequence comprises the nucleic acid sequence of SEQ ID NO: 2. 
     
     
         48 . The method of  claim 8 , wherein the snoRNA sequence is a C/D Box snoRNA sequence, an H/ACA Box snoRNA sequence, a composite C/D Box and H/ACA Box snoRNA sequence, or an orphan snoRNA sequence.