Mitochondrial targeted rna expression system and use thereof

Abstract

Described herein is a mitochondrial-targeted RNA expression system (mtTRES) for delivery of RNA molecules to mitochondria. mtTRES vectors generate RNAs in vivo that are un-capped, non-polyadenylated, and actively directed to mitochondria. The disclosed vectors are capable of delivering either non-coding RNA molecules or RNA molecules encoding a protein of interest to the mitochondria. In particular, the disclosed vectors include (1) an RNAPIII initiation (promoter) sequence, (2) a non-coding leader sequence (NCL), (3) a mitochondrial translation initiation sequence and an ORF encoding a protein of interest, or a sequence encoding a non-coding RNA, and (4) an RNAPIII termination sequence.

1 . A vector comprising in the 5′ to 3′ direction:
 an RNA polymerase III (RNAPIII) promoter sequence; 
 a non-coding mitochondrial leader sequence; 
 a mitochondrial translation initiation sequence and an open reading frame (ORF) encoding a protein, or a sequence encoding a non-coding RNA molecule capable of inhibiting translation of a mitochondrial mRNA molecule; and 
 an RNAPIII termination sequence. 
 
     
     
         2 . The vector of  claim 1 , wherein the RNAPIII promoter sequence is a 5S rRNA RNAPIII promoter sequence or a U6 RNAPIII promoter sequence. 
     
     
         3 . The vector of  claim 2 , wherein the 5S rRNA RNAPIII promoter sequence is at least 95% identical to SEQ ID NO: 9 or the U6 RNAPIII promoter sequence is at least 95% identical to SEQ ID NO: 10. 
     
     
         4 . The vector of  claim 1 , wherein the RNAPIII termination sequence is a 5S rRNA RNAPIII termination sequence. 
     
     
         5 . The vector of  claim 4 , wherein the 5S rRNA RNAPIII termination sequence is at least 95% identical to SEQ ID NO: 11. 
     
     
         6 . The vector of  claim 1 , wherein the non-coding mitochondrial leader sequence comprises a 5S rRNA leader sequence, an MRP leader sequence or an RNAse P leader sequence. 
     
     
         7 . The vector of  claim 6 , wherein the 5S rRNA leader sequence is at least 95% identical to SEQ ID NO: 2, the MRP leader sequence is at least 95% identical to SEQ ID NO: 4, or the RNAse P leader sequence is at least 95% identical to SEQ ID NO: 6. 
     
     
         8 . The vector of  claim 1 , wherein the mitochondrial translation initiation sequence comprises nucleotides 19-38 of any one of SEQ ID NOs: 16-29, nucleotides 11-30 of any one of SEQ ID NOs: 30-43, or SEQ ID NO: 8. 
     
     
         9 . The vector of  claim 1 , wherein at least one codon of the ORF is modified such that the protein can be translated in the mitochondria but not in the cytosol. 
     
     
         10 . The vector of  claim 9 , wherein the codon of the ORF is modified to contain a premature stop codon if translated in the cytosol and a tryptophan codon if translated in the mitochondria. 
     
     
         11 . The vector of  claim 1 , wherein the ORF encodes a protein encoded by a mitochondrial gene. 
     
     
         12 . The vector of  claim 1 , wherein the ORF encodes a reporter protein, and wherein at least one codon of the ORF is modified such that the reporter protein is translated in the mitochondria but not in the cytosol. 
     
     
         13 . The vector of  claim 1 , wherein the vector encodes a non-coding RNA molecule that is capable of inhibiting translation of a mitochondrial mRNA molecule. 
     
     
         14 . The vector of  claim 13 , wherein the non-coding RNA specifically hybridizes with a translation initiation site of the mRNA molecule. 
     
     
         15 . An isolated host cell comprising the vector of  claim 1 . 
     
     
         16 . A method of targeting a recombinant RNA molecule to the mitochondria of a cell, comprising contacting the cell with the vector of  claim 1 , wherein expression of the vector in the cell produces the recombinant RNA molecule which is targeted to the mitochondria. 
     
     
         17 . A method of treating a disease caused by a mutation in a mitochondrial gene, comprising selecting a subject with a disease caused by the mutation in the mitochondrial gene and administering to the subject a therapeutically effective amount of the vector of  claim 1 . 
     
     
         18 . The method of  claim 17 , wherein the subject is administered a first vector and a second vector, wherein the first vector comprises a mitochondrial translation initiation sequence and an ORF encoding a protein, and the second vector comprises a sequence encoding a non-coding RNA molecule capable of inhibiting translation of a mitochondrial mRNA molecule. 
     
     
         19 . The method of  claim 18 , wherein the disease is caused by a mutation in the ATP6 gene, and wherein the ORF of the first vector encodes a wild-type ATP6 protein and the non-coding RNA molecule of the second vector inhibits translation of mutant ATP6 mRNA. 
     
     
         20 . The method of  claim 18 , wherein the first vector comprises one or more silent mutations in the ORF such that translation of the protein is not inhibited by the non-coding RNA molecule of the second vector.