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 . A method of targeting mRNA expressed in the nuclear compartment of a mammalian cell to mitochondrion-bound polysomes said mammalian cell, the method comprising transfecting said mammalian cell with an expression vector,
 wherein said vector comprises:   (i) a mitochondrion-targeting nucleic acid sequence (MTS);   (ii) a nucleic acid sequence encoding said protein in accordance with a universal genetic code (CDS); and   (iii) a 3′UTR nucleic acid sequence, located 3′ of said CDS,   wherein,   said MIS comprises a cDNA sequence of a MIS of a nuclear encoded mitochondrion-targeted mRNA,   said 3 UTR nucleic acid sequence comprises a 3′UTR sequence of a nuclear-encoded mitochondrion-targeted mRNA,   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 MTS and said 3′ UTR nucleic acid sequence are configured to target mRNA expressed by said expression vector in the nuclear compartment to the mitochondrion-bound polysomes of said mammalian cell.   
     
     
         2 . The method of  claim 1 , wherein said MTS nucleic acid sequence comprises the MTS nucleic acid sequence of SOD2, COX10, ACO2, ATP5b, UQCRFS 1, NDUFV 1, NDUFV2, or ALDH2. 
     
     
         3 . The method of  claim 1 , wherein said MTS nucleic acid sequence comprises the MTS nucleic acid sequence of COX10. 
     
     
         4 . The method of  claim 1 , wherein said MTS nucleic acid sequence codes for a peptide selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 46. 
     
     
         5 . The method of  claim 1 , wherein said MTS nucleic acid sequence codes for the peptide of SEQ ID NO: 46. 
     
     
         6 . The method of  claim 1 , wherein said 3′UTR nucleic acid sequence comprises the 3′UTR sequence of SOD2, COX10, ACO2, ATP5b, UQCRFSI, NDUFVI, NDUFV2, ALDH2, or AK2. 
     
     
         7 . The method of  claim 1 , wherein said 3′UTR nucleic acid sequence comprises the 3′UTR sequence of COX10. 
     
     
         8 . The method of  claim 1 , wherein said 3′UTR nucleic acid sequence comprises the nucleic acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 57, and SEQ ID NO:59. 
     
     
         9 . The method of  claim 1 , wherein said 3′UTR nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 59. 
     
     
         10 . The method of  claim 1 , wherein said MTS nucleic acid sequence comprises the MTS nucleic acid sequence of COX10, and said 3′UTR nucleic acid sequence comprises the 3′ UTR nucleic acid sequence of COX10. 
     
     
         11 . The method of  claim 1 , wherein said nucleic acid sequence encoding said protein (CDS) encodes a naturally-occurring mitochondrial protein. 
     
     
         12 . The method of  claim 11 , wherein said nucleic acid sequence is recoded in accordance with the universal genetic code. 
     
     
         13 . The method of  claim 11 , wherein said naturally-occurring mitochondrial protein is selected from the group consisting of ATP6, ND1, and ND4. 
     
     
         14 . The method of  claim 11 , wherein said nucleic acid sequence encoding the naturally-occurring mitochondrial protein is selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 28, and SEQ ID NO: 27. 
     
     
         15 . The method of  claim 1 , wherein said mammalian cell is a human cell.