Importation of mitochondrial protein by an enhanced allotopic approach

Abstract

An expression vector containing appropriate mitochondrion-targeting sequences (MTS) and appropriate 3′UTR sequences provides efficient and stable delivery of a mRNA encoding a protein (CDS) to the mitochondrion of a mammalian cell. The MTS and 3′UTR sequences guide the CDS mRNA from the nuclear compartment of the cell to mitochondrion-bound polysomes, where the CDS is translated. This provides an efficient translocation of a mature functional protein into the mitochondria. A method of targeting mRNA expressed in the nuclear compartment of a mammalian cell to the mitochondrion is also provided. The vector and methods can be used to treat defects in mitochondrial function.

1 . An expression vector configured for the efficient and stable delivery of a protein into the mitochondrion of a mammalian cell, the vector comprising:
 a mitochondrion-targeting nucleic acid sequence (MTS);   a nucleic acid sequence encoding said protein in accordance with a universal genetic code (CDS); and   a 3′UTR nucleic acid sequence, located 3′ of said CDS, configured to target mRNA expressed by the expression vector in the nucleus to the mitochondrion-bound polysomes of the mammalian cell,   wherein,   said MTS consists of a nucleic acid sequence encoding the peptide SEQ ID NO: 46, and   said 3′UTR nucleic acid sequence consists of a conservative variant or fragment of the nucleic acid sequence SEQ ID NO: 47, said conservative variant or fragment still allowing for the mitochondrial targeting of the mRNA.   
     
     
         2 . The expression vector of  claim 1 , wherein the CDS nucleic acid sequence encodes the NADH-ubiquinone oxireductase subunit 4 protein (ND4). 
     
     
         3 . The expression vector of  claim 1 , wherein the nucleic acid sequence encoding the peptide SEQ ID NO: 46 is SEQ ID NO: 30. 
     
     
         4 . The expression vector of  claim 2 , wherein the CDS nucleic acid sequence encoding ND4 is SEQ ID NO: 29. 
     
     
         5 . The expression vector of  claim 1 , wherein said mammalian cell is a human cell. 
     
     
         6 . The expression vector of  claim 1 , wherein the nucleic acid sequence encoding said protein has been optimized with preferred human codon usage. 
     
     
         7 . The expression vector of  claim 2 , wherein the nucleic acid sequence encoding the ND4 protein has been optimized with preferred human codon usage. 
     
     
         8 . The expression vector of  claim 1 , wherein the CDS nucleic acid sequence encodes a protein selected from the group consisting of mitochondrially encoded ATP synthase 6 (ATP6), mitochondrially encoded NADH dehydrogenase subunit 1 (ND1), and NADH-ubiquinone oxidoreductase subunit 4 (ND4). 
     
     
         9 . The expression vector of  claim 8 , wherein the CDS nucleic acid sequence encoding said protein has been optimized with preferred human codon usage. 
     
     
         10 . The expression vector of  claim 8 , wherein the CDS nucleic acid sequence is selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 28, and SEQ ID NO: 27. 
     
     
         11 . The expression vector of  claim 1 , wherein said 3′UTR nucleic acid sequence consists of the nucleic acid sequence SEQ ID NO: 47. 
     
     
         12 . The expression vector of  claim 1 , wherein said 3′UTR nucleic acid sequence consists of a conservative variant of the nucleic acid sequence SEQ ID NO: 47, said conservative variant still allowing for the mitochondrial targeting of the mRNA. 
     
     
         13 . The expression vector of  claim 1 , wherein said 3′UTR nucleic acid sequence consists of a fragment of the nucleic acid sequence SEQ ID NO: 47, said fragment still allowing for the mitochondrial targeting of the mRNA.