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 - 5 . (canceled) 
     
     
         6 . An expression vector configured for the efficient and stable delivery of mitochondrial protein ND4 into the mitochondrion of a mammalian cell, the vector comprising:
 a mitochondrion-targeting nucleic acid sequence (MTS);   a nucleic acid sequence encoding mitochondrial protein ND4 in accordance with a universal genetic code (CDS); and   a 3′UTR nucleic acid sequence. and   wherein,   said MTS comprises a nucleic acid sequence encoding the peptide SEQ ID NO: 46; and   said mitochondrial protein ND4 protein is the protein encoded by SEQ ID NO: 29.   
     
     
         7 . The expression vector of  claim 6 , wherein the MTS is SEQ ID NO: 30. 
     
     
         8 . The expression vector of  claim 6 , wherein the CDS nucleic acid sequence encoding mitochondrial protein ND4 is SEQ ID NO: 29. 
     
     
         9 . The expression vector of  claim 6 , wherein the 3′UTR nucleic acid sequence is the COX10 3′UTR nucleic acid sequence. 
     
     
         10 . The expression vector of  claim 9 , wherein the COX10 3′UTR nucleic acid sequence comprises SEQ ID NO.: 47. 
     
     
         11 . The expression vector of  claim 6 , wherein said mammalian cell is a human cell. 
     
     
         12 . The expression vector of  claim 6 , wherein the MTS comprises nucleotides 7 to 90 of SEQ ID NO: 25. 
     
     
         13 . The expression vector of  claim 6 , wherein the MTS comprises nucleotides 7 to 90 of SEQ ID NO: 26. 
     
     
         14 . The expression vector of  claim 6 , wherein the 3′UTR nucleic acid sequence is the cytosolic SV40 3′UTR nucleic acid sequence. 
     
     
         15 . The expression vector of  claim 14 , wherein the SV40 3′UTR nucleic acid sequence comprises nucleotides 1167 to 1552 of SEQ ID NO: 23. 
     
     
         16 . The expression vector of  claim 14 , wherein the SV40 3′UTR nucleic acid sequence comprises nucleotides 1588 to 1973 of SEQ ID NO: 24. 
     
     
         17 . An engineered mammalian cell which has been transduced, infected and/or transfected by the expression vector according to  claim 6 , wherein the cell is chosen from a bone-marrow cell, a clonal cell, a germ-line cell, a post-mitotic cell, a neuronal cell, a retinal ganglion cell, a progenitor cell; or a stem cell. 
     
     
         18 . A pharmaceutical composition comprising the expression vector according to  claim 6 . 
     
     
         19 . A nucleic acid construct comprising:
 a mitochondrion-targeting nucleic acid sequence (MTS); and   a nucleic acid sequence encoding mitochondrial protein ND4 in accordance with a universal genetic code (CDS);   wherein,   said MTS comprises a nucleic acid sequence encoding the peptide SEQ ID NO: 46; and   said mitochondrial protein ND4 protein is the protein encoded by SEQ ID NO: 29.   
     
     
         20 . The nucleic acid construct of  claim 19 , wherein the MTS is SEQ ID NO: 30. 
     
     
         21 . The nucleic acid construct of  claim 19 , wherein the CDS nucleic acid sequence encoding ND4 is SEQ ID NO: 29. 
     
     
         22 . The nucleic acid construct of  claim 19 , wherein said mammalian cell is a human cell. 
     
     
         23 . The nucleic acid construct of  claim 19 , wherein the MTS comprises nucleotides 7 to 90 of SEQ ID NO: 25. 
     
     
         24 . The nucleic acid construct of  claim 19 , wherein the MTS comprises nucleotides 7 to 90 of SEQ ID NO: 26. 
     
     
         25 . A pharmaceutical composition comprising the nucleic acid construct according to  claim 19 .