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 . A method of allotopically expressing functional ND4 protein in a patient with Leber's hereditary optic neuropathy (LHON), comprising administering to the patient an adeno-associated virus (AAV) vector comprising a recombinant nucleic acid 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,   wherein:
 the MTS sequence comprises a nucleic acid sequence encoding the peptide SEQ ID NO: 46; 
 the mitochondrial protein ND4 protein is the protein encoded by SEQ ID NO: 29; and 
 the MTS sequence allows mitochondrial translocation of the mitochondrial protein ND4. 
   
     
     
         7 . The method of  claim 6 , wherein the MTS is SEQ ID NO: 30. 
     
     
         8 . The method of  claim 6 , wherein the MTS comprises nucleotides 7 to 90 of SEQ ID NO. 25. 
     
     
         9 . The method of  claim 6 , wherein the MTS comprises nucleotides 7 to 90 of SEQ ID NO. 26. 
     
     
         10 . The method of  claim 6 , wherein the 3′UTR nucleic acid sequence is the cytosolic SV40 3′UTR nucleic acid sequence. 
     
     
         11 . The method of  claim 10 , wherein the SV40 3′UTR nucleic acid sequence comprises nucleotides 1167 to 1552 of SEQ ID NO: 23. 
     
     
         12 . The method of  claim 10 , wherein the SV40 3′UTR nucleic acid sequence comprises nucleotides 1588 to 1973 of SEQ ID NO: 24. 
     
     
         13 . The method of  claim 6 , wherein the 3′UTR nucleic acid sequence is the COX10 3′UTR nucleic acid sequence. 
     
     
         14 . The method of  claim 13 , wherein the COX10 3′UTR nucleic acid sequence comprises SEQ ID NO: 47.