RNA TARGETING OF MUTATIONS VIA SUPPESSOR 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 . A method of increasing muscular strength in a subject having a truncated protein associated with DMD, the method comprising administering to the subject a vector encoding two copies of an engineered serine suppressor, wherein the engineered serine suppressor comprises an engineered loop configured to recognize and read through an ochre (UAA) stop signal of a messenger template encoding the truncated protein associated with DMD, thereby increasing muscular strength in the subject; wherein 8 weeks following the administering of the vector to an mdx mouse:
 (i) a neuronal nitric oxide synthase (nNOS) binding site absent from the truncated protein associated with DMD prior to the administering is restored;   (ii) the activity of the nNOS is restored at a sarcolemma of the mdx mouse, wherein the nNOS activity was absent in the sarcolemma of the mdx mouse prior to the administering; and   (iii) a level of full-length protein in the mdx mouse is increased, relative to a level of full-length protein prior to the administering.   
     
     
         2 . The method of  claim 1 , wherein the engineered serine suppressor is charged with a serine. 
     
     
         3 . The method of  claim 1 , wherein the engineered serine suppressor comprises a length of from about 10 nucleotides to about 76 nucleotides. 
     
     
         4 . The method of  claim 1 , wherein an expression of each copy of the engineered serine suppressor is driven by a U6 promoter. 
     
     
         5 . The method of  claim 4 , wherein at least one copy of the engineered serine suppressor is driven by a mouse U6 promoter. 
     
     
         6 . The method of  claim 4 , wherein at least one copy of the engineered serine suppressor is driven by a human U6 promoter. 
     
     
         7 . The method of  claim 4 , wherein a first copy of the engineered serine suppressor is driven by a mouse U6 promoter and a second copy of the engineered serine suppressor is driven by a human U6 promoter. 
     
     
         8 . The method of  claim 1 , wherein the subject is a pediatric human. 
     
     
         9 . The method of  claim 1 , wherein the administering is parenteral. 
     
     
         10 . The method of  claim 1 , wherein the administering is via injection into a tibalis anterior of the subject. 
     
     
         11 . The method of  claim 1 , wherein the administering is via injection into a gastrocnemius of the subject. 
     
     
         12 . The method of  claim 1 , wherein the vector is administered as a unit dose. 
     
     
         13 . The method of  claim 1 , wherein the engineered serine suppressor comprises at least 95% sequence identity to SEQ ID NO: 61, wherein the NNN of SEQ ID NO: 61 is TTA. 
     
     
         14 . The method of  claim 1 , wherein the engineered serine suppressor comprises at least 99% sequence identity to SEQ ID NO: 61, wherein the NNN of SEQ ID NO: 61 is TTA. 
     
     
         15 . The method of  claim 1 , wherein the engineered serine suppressor is SEQ ID NO: 61, wherein the NNN of SEQ ID NO: 61 is TTA. 
     
     
         16 . A method of treating Rett Syndrome in a subject, the method comprising administering to the subject a vector encoding an engineered arginine suppressor, wherein the engineered arginine suppressor comprises an engineered loop configured to recognize and read through an opal (UGA) stop signal of a messenger template encoding a truncated protein associated with Rett Syndrome, thereby treating the Rett Syndrome in the subject. 
     
     
         17 . The method of  claim 16 , wherein the engineered arginine suppressor is charged with an arginine. 
     
     
         18 . The method of  claim 16 , wherein the engineered arginine suppressor is SEQ ID NO:
 63, wherein the NNN of SEQ ID NO: 63 is TCA.   
     
     
         19 . The method of  claim 16 , wherein the engineered arginine suppressor comprises the polynucleotide sequence AATGGATA. 
     
     
         20 . The method of  claim 16 , wherein the administering is parenteral.