RNA TARGETING OF MUTATIONS VIA SUPPRESSOR tRNAs AND DEAMINASES

Abstract

Aspects of the disclosure relate to a gene therapy approach for diseases, disorders, or conditions caused by mutation in the stop codon utilizing modified tRNA. At least 10-15% of all genetic diseases, including muscular dystrophy (e.g. Duchene muscular dystrophy), some cancers, beta thalassemia, Hurler syndrome, and cystic fibrosis, fall into this category. Not to be bound by theory, it is believed that this approach is safer than CRISPR approaches due to minimal off-target effects and the lack of genome level changes.

What is claimed is: 
     
         1 . An adeno-associated viral (AAV) vector plasmid, wherein the AAV vector plasmid encodes an adenosine deaminase acting on RNA (ADAR)-based editing system comprising a guide RNA sequence, wherein:
 the guide RNA sequence comprises a targeting domain with a length of from 60 nucleotides to 100 nucleotides and, upon hybridization to a target RNA, the targeting domain and the target RNA form a mismatch corresponding to a cytosine of the targeting domain and an adenosine of the target RNA, wherein the mismatch is centrally positioned within the targeting domain;   the guide RNA sequence does not comprise a chemical modification;   upon administration of the vector to a human subject, the guide RNA sequence when hybridized to the target RNA recruits an adenosine deaminase enzyme endogenous to the human subject that performs a targeted chemical modification on the adenosine of the target RNA; and   the ADAR-based editing system does not comprise an ADAR1 or ADAR2 enzyme encoded by the AAV vector plasmid.   
     
     
         2 . The AAV vector of  claim 1 , wherein the adenosine deaminase enzyme endogenous to the human subject is an endogenous ADAR polypeptide. 
     
     
         3 . The AAV vector of  claim 2 , wherein the endogenous ADAR polypeptide is an ADAR1 polypeptide. 
     
     
         4 . The AAV vector of  claim 2 , wherein the endogenous ADAR polypeptide is an ADAR2 polypeptide. 
     
     
         5 . The AAV vector of  claim 1 , wherein the AAV vector plasmid is an AAV2 vector plasmid, an AAV5 vector plasmid, an AAV8 vector plasmid, or an AAV9 vector plasmid. 
     
     
         6 . The AAV vector of  claim 1 , wherein the targeting domain comprises at least about 80% sequence homology to any one of SEQ ID NO: 182-206. 
     
     
         7 . The AAV vector  claim 1 , wherein the target RNA is mRNA or pre-mRNA. 
     
     
         8 . The AAV vector of  claim 1 , wherein the target RNA comprises the adenosine is a point mutation implicated in a disease, disorder, or condition in a subject. 
     
     
         9 . The AAV vector of  claim 8 , wherein the point mutation is a non-sense mutation, a missense mutation, or a splice site. 
     
     
         10 . The AAV vector of  claim 9 , wherein the disease comprises muscular dystrophy. 
     
     
         11 . The AAV vector of  claim 1 , wherein the target RNA does not comprise a non-sense mutation or a missense mutation. 
     
     
         12 . The AAV vector of  claim 1 , wherein administration of the vector to the human subject results in an effective amount of the guide RNA sequence to treat a disease or condition. 
     
     
         13 . The AAV vector of  claim 1 , wherein the targeting domain comprises a length of about 100 nucleotides. 
     
     
         14 . The AAV vector of  claim 1 , wherein the AAV vector contains a promoter comprising a human U6 promoter, a mouse U6 promoter, a CMV promoter, or any combination thereof. 
     
     
         15 . A kit comprising the AAV vector of  claim 1  in a container. 
     
     
         16 . A method of treating a disease or condition in a subject in need thereof comprising:
 administering the AAV vector of  claim 1  to the subject in need thereof, wherein the disease or condition is a muscular dystrophy, ornithine transcarbamylase deficiency, Nougaret night blindness, Usher syndrome, Parkinson disease, Wilson disease, hereditary tyrosinemia, cancer, beta thalassemia, Hurler syndrome, or cystic fibrosis.