Importation of mitochodrial 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,   wherein,   said MTS comprises a nucleic acid sequence encoding the peptide SEQ ID NO: 46,   said 3′UTR nucleic acid sequence comprises the nucleic acid sequence SEQ ID NO: 47,   said vector does not comprise: any sequence identical to a 3′UTR of a naturally occurring mRNA which is a nuclear-transcribed but not mitochondrion-targeted mRNA, a cDNA sequence of said 3′UTR of a naturally occurring mRNA, or a DNA sequence coding for said 3′UTR of a naturally-occurring mRNA in accordance with the universal genetic code, and   said vector does not utilize a post-translation importation pathway, but utilizes a co-translation importation pathway from the nucleus to the mitochondrion.   
     
     
         2 . The expression vector of  claim 1 , wherein the CDS nucleic acid sequence encodes naturally-occurring functional mitochondrial 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.